Genome stitched together by hand
Scientists construct building blocks for artificial life.
Scientists have succeeded in stitching together an entire bacterial genome, creating in the lab the full set of instructions needed to make a living thing. The stage is now set for the creation of the first artificial organism — and it could be achieved within the year.
The genome for the pathogenic bacterium Mycoplasma genitaliumwas made in the laboratory by Hamilton Smith and his colleagues at the J. Craig Venter Institute in Rockville, Maryland. The genome has 582,970 of the fundamental building blocks of DNA, called nucleotide bases, making it more than a factor of ten longer than the previous-longest stretch of genetic material created by chemical means.
Now the team at the Venter institute, which includes the institute’s founder, genomics pioneer Craig Venter, will aim to discover whether cells can be ‘booted up’ into action when loaded with this genetic programme. “This is the next step and we are working on it,” says Smith.
Venter and his colleagues have already managed to transplant the DNA from one bacteria into another, making it change species (see Genome transplant makes species switch). These bacteria were closely related to M. genitalium.If the transplant can be repeated with a man-made genome adapted from M. genitalium, the result could qualify as the first artificial life form (see '').
DNA is synthesized by sequentially adding one of the four nucleotide bases (denoted A, T, G and C) to a growing chain in a specified sequence. It is beyond current capabilities to join up half a million or so bases in a single, continuous process — the strand becomes unstable and breaks. So the researchers ordered 101 custom-made fragments or ‘cassettes’, each of about 5,000–7,000 bases each, from companies Blue Heron Biotechnology in Bothell, Washington; DNA2.0 in Menlo Park, California; and GENEART of Toronto, Ontario. These were designed with overlapping sequences so they could be stuck together later by enzymes.
To distinguish the synthetic DNA from the genomes of ‘wild’ M. genitalium,Smith and his colleagues included ‘watermark’ sequences — stretches of DNA that designate its artificiality. These watermarks are inserted at sites in the genome that can tolerate such additions without their genetic function being impaired. The researchers made one further change to the natural genome: they altered one gene in a way that was known to render M. genitaliumunable to stick to mammalian cells. This ensured that cells carrying the artificial genome could not act as pathogens. The revised sequence will be published in GenBank, they say.
The cassettes were then stitched together into strands that each contained one-quarter of the total genome using DNA-linking enzymes within cells of the bacterium Escherichia coli. But, for reasons that the researchers don’t yet understand, the final assembly of these quarter-genomes into a single strand didn’t run smoothly in the bacterium. So the team transferred them into cells of brewers’ yeast to carry out the final steps of the assembly.
Smith and his colleagues then extracted the synthetic genomes from the yeast cells, using enzymes to chew up the yeast’s own DNA. They finally checked the sequence of the remaining DNA to verify that it matched what they were aiming to make.
Advances in DNA synthesis might ultimately make it possible to simply construct the entire genome in a single effort. But Dorene Farnham, director of sales and marketing at Blue Heron, stresses that this wouldn’t solve all the problems. Even if a long string of DNA could be made in the lab, it could fall apart once stuck into a cell, she says. “There are many other factors that go into getting these synthetic genes to survive in cells,” she says.
Great things, small packages
M. genitaliumhas the smallest genome known of any organism that can grow and replicate independently. Its DNA contains the instructions for making just 485 proteins, which orchestrate the cells’ functions.
Its small genome makes M. genitaliuma candidate for the basis of a ‘minimal organism’, which would contain the bare minimum of genes needed to survive. The Venter institute team thinks that around 100 of the bacterium’s genes aren't strictly necessary for life — but they don’t know which 100.
The way to test that would be to make versions of the M. genitalium genome that lack some genes, and see whether it still provides a viable ‘operating system’ for the organism.
The team hopes that a stripped-down version of the M. genitalium genome might serve as a general-purpose chassis to which might be added all sorts of useful designer functions, such as genes that turn the bacteria into biological factories for making carbon-based ‘green’ fuels or hydrogen when fed with nutrients.
The next step towards that goal is to build potential minimal genomes from scratch, transplant them into Mycoplasma, and see whether the cells survive. “We plan to start removing putative ‘non-essential’ genes and test whether we get viable transplants,” says Smith.
- Gibson, D. G. et al. Science Express doi:10.1126/science.1151721 (2008).