In preliminary proof-of-concept studies, Church says, his team has “changed every codon that can be changed” in a collection of essential (mostly ribosomal) genes, partially freeing up a dozen or so codons along the way.
Finding the starting point
The key to a successful MAGE experiment is knowing where to direct your oligos. Sometimes the targets are obvious: hit every promoter in a given pathway, say, to optimize that biochemical process. Other times, though, it isn't clear what genes might make the most effective targets.
“Given an impossibly large combinatorial search space, how do you migrate across that in the most efficient manner and on a laboratory time-scale?” asks Ryan Gill, associate professor of chemical and biological engineering at the University of Colorado.
This is where trackable multiple recombineering (TRMR) comes in. Invented by Gill and former postdoc Joe Warner, TRMR uses recombineering — the ability to introduce single-stranded DNA into cells which is then incorporated into the genome — to create libraries of cells in which every promoter is tagged with a trackable barcode and turned either up or down, some 8000 clones in all (4).
Prior to initiating the experiment, researchers sequence the pool to capture the library's barcodes, providing an indication of the abundance of each clone. Then, cells are exposed to whatever condition it is the researcher wants to optimize.
Suppose, for instance, that you want to create a strain that is tolerant of high concentrations of alcohol. “You can read the literature as much as you want, you're not going to find out, oh here's the magic recipe, here are the 10 genes that if you turn them on in this particular combination of expression levels, you're going to get ethanol tolerance at 80 gm/L,” explains Gill.
But by exposing the TRMR library to increasing concentrations of alcohol and identifying the clones that thrive or die off, you can get a sense of where to start.
Gill, who calls TRMR “a search technology” for genome engineers, says the technique helps address the oft-repeated criticism of genome engineering, that researchers know how to write DNA, but not what to write. Yet pairing the technique with MAGE is complicated, notes Gill, because mutations that boost growth in isolation (as in TRMR) don't necessarily perform well in combination.
“Sometimes there can be antagonistic interactions, so that one plus one does not equal two, it actually equals point-seven,” explains Gill. “It makes [the modified genome] worse than the original one.”
Not all bioengineers want to retool an entire genome. Many focus on the comparatively easier problem of pathway engineering, turning bacteria into microbial factories for plant metabolites, pharmaceuticals, for instance.
MIT biologist Anthony Sinskey, along with research scientist Christopher Brigham and chemistry grad student Jingnan Lu, are exploring the possibility of turning sugars, organic acids, and other carbon sources into gasoline using a microbe called Ralstonia eutropha. Under stress conditions Ralstonia very efficiently stores carbon as a polymer called PHB, which is chemically similar to petroleum. Another huge positive, Ralstonia is a voracious and indiscriminate consumer of carbon. “It's got a wide range of things that it can use for food, and a lot of those things can be found in waste streams,” says Brigham.
Brigham and Lu's idea was to force the cells to turn that waste into gasoline by redirecting intermediates from the branched chain amino acid biosynthetic pathway. First though, they had to eliminate the organism's ability to store carbon in the form of PHB, and add a missing enzyme called KIVD (ketoisovalerate decarboxylase), which converts leucine and valine precursors into aldehydes, the penultimate step in creating gasoline in the cell.
Ralstonia doesn't have a KIVD gene, so the team added one from Lactococcus. But simply adding a single gene that wasn't going to be enough; they also had to turn on an endogenous alcohol dehydrogenase gene and boost expression of other members of the branched chain amino acid biosynthetic pathway. They deleted a few “carbon sink” pathways for good measure.