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Sensors for All

A versatile modular strategy for detecting small molecules in eukaryotes

May 1, 2016
Ruth Williams

SENSOR SET-UP: To detect a small molecule of interest (the ligand), a conditionally stable ligand-binding domain (LBD) is fused to a reporter, such as green fluorescent protein (GFP). The complex degrades if the ligand is not present (1), and activates the reporter when it is (2). In another demonstration of this sensor, researchers connected the LBD to a DNA-binding domain (DBD) (3). When the ligand is present, the DBD hooks onto to a site in the genome (red), which results in the expression of a specified reporter gene (yellow) (4).© GEORGE RETSECK; ELIFE, 4:E10606, 2015

The ability to detect small molecules of interest has wide applicability in biological research, biotechnology, and especially synthetic biology. For example, turning cells into factories that produce small molecules—for use as drugs, biofuels, and more—is the goal of many synthetic biology endeavors. Just like regular factories, cellular ones require optimization. “In many cases we can create a valuable compound, but at a very low yield,” says Dan Mandell, a postdoctoral researcher in George Church’s Harvard University lab.

Scientists can attempt to improve production, but there is often no fast way to know whether they’ve succeeded. Mass spectrometry, for example, is a very sensitive and reliable way to detect small molecule production, says Mandell, but it’s “somewhat cumbersome, expensive, and slow.”

Specific sensors exist for only a handful of compounds. But now, Mandell and colleagues have devised a system that, in theory, could be used to make sensors for essentially any small molecule and that can be modified for use in any cell.

The key to the system is to create a conditionally stable ligand-binding domain (LBD), a peptide that tightly binds the small molecule in question, but that rapidly degrades without it. This LBD can then be fused to a range of proteins—ones that fluoresce, ones that drive transcription of reporter genes, and so on—such that the presence of the small molecule leads to the production of an easily detected signal.

So far the team has created two LBDs—for digoxin and progesterone—and fused them to a variety of proteins to produce a range of sensors that can detect these two steroids in yeast, human cells, and even plants. “Creating sensors to detect and measure the levels of molecules inside the cell is a holy grail for synthetic biology,” explains Jay Keasling of the University of California, Berkeley. (eLife, 4:e10606, 2015)

SMALL MOLECULE DETECTION HOW IT WORKS PROS CONS
Mass spectrometry Contents of cells are ionized, accelerated through a mass spectrometer, and the small molecule of interest is detected and quantified. Highly sensitive, well-established, no up-front engineering of proteins required Very low throughput (a few dozen samples per machine per day), making optimization of production extremely slow
Conditionally stable LBD biosensors The LBD is rapidly degraded in the absence of the molecule of interest, but is stabilized in its presence, enabling the activation of a fused reporter domain. The more abundant the molecule, the stronger the reporter signal. Massively high throughput: billions of yeast cell variants or millions of mammalian cells can be assessed per day.

Reporter domains can be changed depending on the cell system
and desired readout.

 

A highly specific conditionally stable LBD must exist naturally or be engineered for each small molecule of interest.

 

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