A group of scientists have developed a new cell-programming method called Multiplex Automated Genome Engineering (MAGE) that allowed them to edit multiple genes in parallel instead of targeting one gene at a time. They were able to transform E. coli cells into efficient factories in three days, a feat that would take most biotech companies months or years, according to the team.
“The goal was to use information gleaned from genetics and genomics to rapidly engineer new functions and improve existing functions in cells,” remarks postdoctoral researcher Farren Isaacs, Ph.D. Dr. Isaacs is also one of the first authors on the paper titled “Programming cells by multiplex genome engineering and accelerated evolution,” which appeared online in Nature on July 26.
The researchers selected a harmless strain of the intestinal nemesis E. coli and added a few genes to its solitary circular chromosome. This coaxed the organism to produce lycopene, a powerful antioxidant that occurs naturally in tomatoes and other vegetables. They were then able to focus on tweaking the cells to increase the yield of this compound.
For this step, the scientists wanted to move away from using recombinant DNA technology, or gene cloning, a technique that involves isolating, breaking up, reassembling, and then reinserting genes.
“Genes function in teams, not in isolation,” says graduate student Harris Wang, the paper's other first author. “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.”
So the team generated genetic diversity at an unprecedented rate, thus increasing the odds of finding cells with desirable properties. The E. coli bacterium contains approximately 4,500 genes. The team focused on 24 of these, honing a pathway with potential to increase production of the antioxidant and 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. They were 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 and some 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 these steps.
“We accelerated evolution, generating as many as 15 billion genetic variants in three days and increasing the yield of lycopene by 500 percent,” Wang says. “Can you imagine how long it would take to generate 15 billion genetic variants with traditional cloning techniques? It would take years.”
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.