ASU Research E-Magazine: Hunting for Mutants

Scott Bingham is a hunter. His quarry is mutant DNA. His hunting ground is a petri dish.

Bingham works closely with ASU botanist Andrew Webber. Their quarry of choice is an organism called Chlamydomonas reinhardtii. Lately, his efforts have focused on creating new types of selectable markers, means by which one can ensure that a particular new mutation has taken hold within the target organism. There are many steps to creating a useful selectable markers. One starts with natural selection itself.

“When we try to select the mutant that’s resistant to a compound like norfluorozone (a common herbicide), we can generally put about 10,000,000 cells on one plate, in one little petri dish. And from those 10,000,000 cells we can sometimes pick up 5 or 10 mutants on one plate that might be resistant to a particular compound. That’s basically what we’ve done with Chlamydomonas,” reports Bingham.

Colonies that have resistance are easy to spot.

“If you see something that’s very round, all by itself on a plate, eventually after a week or two, that arose from one single cell that became resistant,” Bingham says. “Then we can just pick them off the plate with a toothpick. We then restreak them again on another plate that would have the same selective agent on it, to make sure that it’s really resistant to the agent that we’ve been using.”

Because DNA does occasionally make copying errors, given 10,000,000 cells, some errors have already occured. The resistance to the herbicide fluorozone turns out to be a single modified base pair in the strand of millions.

This particular mutation is not regularly seen in nature, because it causes somewhat deleterious side effects. But in the harsh environment of a petri dish flooded with norfluorozone, it’s just what Chlamydomonas ordered.

As Bingham notes, this gene, once isolated through a rather arduous series of steps, can be attached to the plasmid vector carrying the new protein sequence one is firing into Chlamydomonas. Thus it becomes possible to selectively kill off those algae that have not received the modification, and through successive generations, breed only those cells with quite a strong resistance, thus, a sizable percentage of chlorophylls with the desired trait. Bingham has also turned his attention and expertise to other organisms.

“We’re also using Chlorella, which is an organism of much more industrial significance. It’s actually used to make many commerical products,” he adds. For example, Chlorella is used to make stable isotopically labeled chemicals.

“You use the the organism to make products for you. For instance, glucose is a sugar. It’s not ordinary glucose because the algae can take up carbon 14, instead of carbon 12,” Bingham explains. Carbon 14 is weakly radioactive, and thus can be traced through organisms that feed on this modified glucose. The creation of herbicide-resistant markers is particularly important in work on plants. Antibiotics are often used as markers in bacteria, but have their drawbacks. Herbicide resistance can be reproduced by Escherichia coli without affecting it, and thus is quite a useful trait to have.—John Svetlik