The Next Step For The Synthetic Genome

Monya Baker, The Next Step For The Synthetic Genome, Nature 473 (2011)

Scientists at the J. Craig Venter Institute in Rockville, Maryland, had chemically synthesized DNA and Placed it inside a bacterial cell emptied of its own genetic material. Tests a few days after the insertion showed that the 1-million-base-pair-long synthetic genome was able to run the cellular machinery.

The story of the field so far is, “can write DNA, nothing to say”, says Drew Endy, a synthetic biologist at Stanford University in California. “We can compile megabases of DNA no one is designing beyond the kilo base scale.” We frankly don’t understand biology well enough to start designing genomes de novo.”

Transplanting DNA molecules into cells is not easy, nor is getting the DNA to ‘boot up’ once it is in place. And because the genomes will be far from perfect, researchers will need ways to tweak and test many variants.

No one expects fast results, and much of the work will be tedious. The Venter Institute spent 15 years and US$40 million creating the technology to build and transplant a genome.

Four species were involved in the genome transplant: Mycoplasma mycoses to provide the source code, Escherichia coli to copy DNA pieces, backer’s yeast (Saccharomyces cerevisiae) to assemble them into a million-base-pair circle and Mycoplasma capricolum  to provide the recipient shell.

Synthetic biology often adopts the language of engineers: rather than talking about genes, networks and biosynthetic pathways, practitioners prefer to talk about parts, devices, modules. ‘Parts’ refer to the protein coding section of a gene and sundry regulatory sequences that tune gene expression. A ‘device’ is an assembly of parts that together perform a particular function, often turning a protein’s production on or off. And a ‘module’ or pathway is a collection of devices that carry out more-complex functions, such as coordinating a chemical synthesis or shunting cells between ‘growth’ and ‘production’ modes.

The Massachusetts Institute of Technology (MIT) in Cambridge maintains a Registry of Standard Biological Parts (http://partsregistry.org) descriptions of these parts are often incomplete, and they don’t all work as described.

To address this issue, Endy and bioengineers from the University of California, Berkeley, launched the International Open Facility Advancing Biotechnology (BioFab) in Emeryville in 2009, with a grant from the National Science Foundation. The BioFab aims to boost the supply of working parts both by optimizing the parts themselves and by developing systems to swiftly design genetic constructs. The BioFab currently provides 350 promoters, grouped into ten levels of protein production.

Researchers can link elements using a system called BioBricks, in which sequences are cut out of circular genetic elements called plasmids by restriction enzymes specific to a particular series of nucleotides at the start and end of the sequence. The desired parts are then stitched together into larger plasmids by other enzymes. Assembled sequences can then be replicated in bacteria.

But BioBricks-type methods are limited by their use of restriction enzymes. Because the enzymes cut DNA whenever they encounter a particular series of nucleotides there are ‘forbidden sequences’ that must be excluded from the genetic construct to avoid errant cutting. The larger a construct becomes, the harder it is to avoid such sequences. To circumvent this problem, researchers have developed assembly ‘overlap’ methods, in which opposite ends of molecules are joined as DNA is copied. Dozens of separate pieces of DNA can be assembled in the same reaction, often totaling a few thousand nucleotides. These methods have their own drawbacks, however. Most copy DNA using the polymerase chain reaction (PCR), which can introduce errors.

Assemblies larger than about 100 kilo bases may be best put together inside cells, because big DNA molecules are fragile and difficult to manipulate. In vitro replication is also less accurate than cell’s machinery.

In 2005, Mitsuhiro Itaya, a biochemist now at keio University in Tsuroka, Japan, and his colleagues constructed a 3,500-kilobases cyanobacterium genome. They cut the genome of the bacterium Synechocystis PCC6803 into large chunks and propagated them in specially prepared plasmids in E. coli. The plasmids were then transferred into a third species, Bacillus subtilis, where the DNA was stitched together.

Assembly methods aren’t interchangeable. Overlap sequences that work for one method often don’t work for others, so researchers who run into problems with one technique have to start from scratch.

If researchers start building genomes or even large part of genomes they will have to think about how the DNA will wrap up on itself, and how they can place genes in chromosomes so that they end up in the right places, says Ellis. “It’s a whole other aspect we’ll have to uncover if we’re going to do genome engineering.

Jef Boeke, a molecular biologist at Jons Hopkins Medical Institue in Baltimore, Maryland, believes that genome-scale engineering is coming more quickly than many think. He is building artificial yeast chromosomes, each about the same size as the M. mycoides genome. Although he hasn’t yet been able to design an entire new genome, he has developed techniques to make systematic alterations in existing genetic codes.

In a colourful demonstration in 2009, Church and his colleagues described a high-throughput editing system. Multiplex-automated genome engineering (MAGE) mixes bacteria with synthesized stretches of DNA that are designed to target many areas in the genome; carefully timed jolts of electricity cause the bacteria to take up the DNA as they grow in culture.

The most difficult problem may well be one of the least discussed: putting the genome to work. Although Itaya has synthesized large genomes inside cells, the introduced genomes do not go on to produce proteins.

In fact, Venter thinks that adapting genomes to work in different cell types may be one of the most difficult tasks. The creation of the first synthetic cell is illustrative: the team had to remove certain enzymes from recipient cells to keep them from cutting up the foreign DNA. And moving to other species is going to be even more difficult. Unlike Mycoplasma, many microbes contain tough cell walls that resist the introduction of DNA.

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