|MAGE enables the rapid and continuous generation of sequence diversity at many targeted chromosomal locations across a large population of cells through the repeated introduction of synthetic DNA. Wang et al., Nature. Click to enlarge.|
A new cell programming method called Multiplex Automated Genome Engineering (MAGE) promises to give biotechnology, in particular synthetic biology, a powerful boost. MAGE was developed by a team led by a pair of researchers in the lab of Harvard Medical School Professor of Genetics George Church. In addition to his scientific accomplishments, Dr. Church co-founded Joule Biotech (solar fuels), LS9 (bio-petroleum), and Knome (full human genome sequencing).
Using the platform, the team rapidly refined the design of a bacterium by editing multiple genes in parallel instead of targeting one gene at a time. They transformed E. coli cells into efficient bio-factories that produced a desired compound in three days—a feat that would take most biotech companies months or years. A paper on their work was published online in the journal Nature on 26 July.
“We initiated the project to close the gap between DNA sequencing technology and cell programming technology,” explains graduate student Harris Wang, the paper’s co-first author. “The goal was to use information gleaned from genetics and genomics to rapidly engineer new functions and improve existing functions in cells,” adds postdoc Farren Isaacs, the other first author. “We wanted to develop a new tool and demonstrate how to apply it; we were determined to hand labs a hammer and a nail.”
While in vitro and directed evolution methods have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes, the team notes. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity.
The MAGE platform allows scientists to break free of linear genetic engineering techniques and move beyond the serial manipulation of single genes.
The researchers selected a strain of E. coli and added a few genes to its solitary circular chromosome to produce lycopene, an antioxidant that occurs naturally in tomatoes and other vegetables. Next, they focused on tweaking the cells to increase the yield of this compound.
Traditionally, labs would accomplish this type of transformation by using recombinant DNA technology, also known as gene cloning, a complicated technique that involves isolating, breaking up, reassembling, and then reinserting genes.
The Church lab researchers took a different approach. “Genes function in teams, not in isolation,” says Wang. “Cloning often encourages us to ignore the interdependence of genes and oversimplify the cellular system. We might forget, for example, that one mutation can strengthen or weaken the effects of another mutation.”
|“We decided to engineer in the context of biology, embracing evolution rather than trying to fit a square peg in a round hole. This automated, multiplex technology will allow labs to engineer entire pathways and genomes and take cell programming to a whole new level.”|
The E. coli bacterium contains approximately 4,500 genes. The team focused on 24 of these—honing a pathway with tremendous potential—to increase production of the antioxidant, optimizing the sequences simultaneously. They took the 24 DNA sequences, divided them up into manageable 90-letter segments, and modified each, generating a suite of genetic variants. Next, armed with specific sequences, the team enlisted a company to manufacture thousands of unique constructs. The team was then able to insert these new genetic constructs back into the cells, allowing the natural cellular machinery to absorb this revised genetic material.
Some bacteria ended up with one construct, some ended up with multiple constructs. The resulting pool contained an assortment of cells, some better at producing lycopene than others. The team extracted the best producers from the pool and repeated the process over and over to further hone the manufacturing machinery. To make things easier, the researchers automated all of these steps. With their accelerated evolution approach, the team generated as many as 15 billion genetic variants in three days, and increased the yield of lycopene by 500%.
The pathway the team refined plays a role in the synthesis of many valuable compounds, ranging from hormones to antibiotics, so the reprogrammed bacteria can be used for a variety of purposes. In addition, the MAGE platform itself unlocks new possibilities.
This research is funded by NSF, DOE, DARPA, the Wyss Institute for Biologically Inspired Engineering, NIH and NDSEG.
Harris H. Wang, Farren J. Isaacs, Peter A. Carr, Zachary Z. Sun, George Xu, Craig R. Forest & George M. Church (2009) Programming cells by multiplex genome engineering and accelerated evolution. Nature doi: 10.1038/nature08187