Making molecules is no easy task. An increasingly attractive approach: have bacteria do the work for us.
Such efforts are part of the growing discipline of synthetic biology, where biological processes are altered to achieve specific goals. In this field, researchers can genetically modify bacteria so they’ll produce a desired molecule – one that can be used to help make medicines or chemical products, for example
This process is far from being perfected, though. A popular approach is to create millions or billions of different mutant bacteria in hopes of finding rare variants that produce more of the desired molecule than their “parents.” This, in turn, presents a classic needle-in-a-haystack challenge. How do you identify the handful of bacteria you want among the huge number you don’t?
Patrick Cirino, an associate professor of chemical and biomolecular engineering with the University of Houston Cullen College of Engineering, has recently published an article in the Journal of the American Chemical Society on the creation of a new biosensor that solves this problem, at least in one case, and shows an approach that can be applied to screening a variety of targeted molecules.
In this case, the group sought bacteria that produce triacetic acid lactone (TAL), a precursor to several chemical processes. But first, a system to screen for TAL production was developed. Cirino’s biosensor for TAL is produced by and lives inside E. coli bacteria. Creating this biosensor is a multistep process that starts by mutating the regulatory protein AraC, which is essentially an on/off switch for certain genes in E. coli. These genes are turned on when AraC binds to a molecule known as arabinose. Cirino’s goal, though, was to create an AraC protein that instead binds with TAL.
To do this, he generated millions of bacteria, each with a different mutated AraC protein. He also tweaked the gene controlled by AraC (or an AraC mutant) so that the cells would produce a fluorescent protein when the AraC switch is flipped. Next, he introduced TAL into the mix.
In most of the mutated bacteria, nothing happened. A few dozen, though, turned fluorescent. The fluorescence signaled the presence of an AraC mutant that was able to respond to TAL. That alone is an important finding, said Cirino. “These particular mutated AraC proteins are now biosensors for TAL. We know this because genes get turned on when TAL is present.”
The creation of a biosensor for TAL didn’t mark the end of this work, though. The next step was creating another batch of mutated E. coli, this time to find ones that produce increased amounts of TAL. First, the TAL-sensing E. coli was endowed with the ability to produce small amounts of TAL by inserting a gene that comes from the plant Gerbera hybrida, a type of daisy. At the same time, Cirino again altered the gene activated by the AraC-TAL mutant. Upon activation the cells produce an enzyme that is easily detected by blue color formation on agar plates (basically petri dishes containing a growth medium for microorganisms). Finally, he created tens of thousands of different variants by mutating the G. hybrida gene, with the goal of finding mutants that generate greater amounts of TAL.
After letting the bacteria grow for several hours, locating the ones that produced the most TAL was simple, Cirino said. He just had to find the ones that had turned the most intensely blue the fastest. Deeper blue E. coli colonies corresponded to increased interactions between TAL and the AraC-based TAL sensor protein, and hence more TAL production.
While being able to generate and sense the creation of large amounts of TAL is nice, Cirino said, it is not the most important aspect of this project. Given the growing importance of synthetic biology, answering fundamental questions, like how to sense the presence of a desired molecule, has great value. “The broader impact is not so much making TAL, but developing and being able to demonstrate an application of a novel small molecule biosensor. That’s the biggest advance, showing we can make and use these customized sensors,” he said.