NEW YORK (GenomeWeb) – Researchers from the J. Craig Venter Institute and Synthetic Genomics have designed and synthesized a bacterium with a genome of just 473 genes.
After failing to create a viable cell by building a minimal genome based on current molecular biology knowledge and transposon mutagenesis, Craig Venter from JCVI and Synthetic Genomics and his colleagues turned to improved transposon mutagenesis to uncover quasi-essential genes — those that are needed for robust growth, though not necessarily life. After adding them back in, the researchers went through additional rounds of design, synthesis, and testing to produce the synthetic Mycoplasma mycoides JCVI-syn3.0, as they reported today in Science.
This minimal bacterial genome includes genes involved in macromolecule synthesis and processing, but also nearly 150 genes of unknown function.
"We have discovered some essential facts of biology by doing this," Venter, the founder and CEO of JCVI and the co-founder and co-chief scientific officer of Synthetic Genomics, said during a press briefing. "[This] means we know about two-thirds of essential biology, [but] we're missing a third, which is a very important lesson."
Venter and his colleagues previously reported their development of a synthetic M. mycoides, dubbed JCVI-syn1.0, which they then demonstrated was functional and self-replicating. In this study, they strove to strip M. mycoides JCVI-syn1.0 down to its bare essentials: just what it needs to live and reproduce in a timely fashion in the lab.
Previous estimates based on comparative genomics and mutagenesis studies pegged the common core of needed genes to be between 256 and 375, the researchers noted. Based on that earlier literature and transposon mutagenesis data, Venter and his colleagues designed an M. mycoides genome that was 483 kilobase pairs in size and included 432 protein-coding genes and 39 RNA genes — some 440 fewer genes than their syn1.0 genome.
In designing this genome, they divvied it up into eight overlapping segments so that each section could be made and tested individually in a background of the seven other segments from the syn1.0 genome. However, only one segment from this initial build yielded viable colonies, and even they did not grow well. This suggested to the researchers that some essential or quasi-essential genes had been tossed out.
To identify those apparently needed genes, the researchers used Tn5 mutagenesis to characterize genes within the syn1.0 genome as essential, non-essential, and quasi-essential —genes that slowed growth when deleted, for instance. The syn1.0 genome included 432 non-essential, 240 essential, and 229 quasi-essential genes.
Based on that and additional deletion data, the investigator re-designed a minimal genome that included additional genes. They again synthesized it as eight segments that they tested in the syn1.0 background. This time, they reported that each of the eight segments yielded a viable colony.
However, when they combined the eight fragments into one genome, it did not produce a viable cell when transplanted into M. capricolum.
In bacteria, Venter and his colleagues noted, multiple genes often cover essential or quasi-essential functions. Because of that, a seemingly non-essential gene could actually be essential, though that would only be revealed if its partner were also removed.
"If you know nothing about airplanes and you're looking at a 777, and you're just trying to find out functions of parts by removing them, and you remove the engine from the right wing, the airplane can still fly and land, so you might say that's a non-essential component," Venter said. "You don't really discover the essentiality until you remove the second one."
Again using Tn5 mutagenesis and deletion studies, they homed in such genes that needed to be added back into their minimal genome.
This syn2.0 genome, they reported, was 576 kilobase pairs in size with 478 protein-coding genes and 38 RNA genes. Venter and his colleagues further subjected syn2.0 to additional rounds of Tn5 mutagenesis and deletion studies to pare its genome down further.
Through this, they generated syn3.0, a 531-kilobase pair genome harboring 438 protein-coding and 35 RNA genes.
Intriguingly, the functions of about a third of the genes contained within this minimal genome are unknown. Most of the genes with known biological functions are involved in gene expression, but the function of 149 genes can't be assigned to a specific function. While some of them are associated with generic functional categories such as membrane transport, 65 genes still have a wholly unknown function.
Cells with such minimal genomes could be used in both basic and applied research, according to Venter and his colleagues. Gene sets, they noted, could be added back into the minimal genome to understand their function. In addition, cells with a minimal genome could serve as a chassis for the industrial production of medicine, biofuels, and more.
JCVI and Synthetic Genomics have filed patent applications regarding this work.