Electronic gizmos and living cells alike can process signals, turning inputs into outputs—even recognizing when gray-area signaling is dark enough, or light enough, to justify decisions that are, well, black and white. In cells, this kind of signal processing is at the root of processes such as differentiation and development. Imagine if one could hack such processes! One could reengineer human cells, guide immune and stem cell functions, and realize novel cell-based therapeutics.

Sounds great, right? There’s a problem, however. Cells have never heard of Radio Shack. That is, they don’t rely on standardized circuit components, much to the frustration of synthetic biologists, for whom the reengineering of cellular circuitry can be as tricky as taking a soldering iron to a printed circuit board. To date, synthetic biologists have been able to reconstruct and mimic simpler forms of cell-based signal processing. But the more complex forms, the forms behind binary-like responses to continuous multivalent signals, has proven elusive.

Nonetheless, analog-to-digital capabilities can be brought to synthetic biology, report scientists representing Rice University, Boston University, MIT, Harvard University, the Broad Institute, and Brandeis University. These scientists used a biochemical process called cooperative assembly to engineer genetic circuits that were able to both decode frequency-dependent signal and conduct dynamic signal filtering. In other words, the genetic circuits could act like a tunable, analog-to-digital converter.

Details of this work appeared April 18 in the journal Science, in an article titled, “Complex signal processing in synthetic gene circuits using cooperative regulatory assemblies.” Cooperative assembly can be programmed, the article suggests, to fine-tune cellular behavior along a digital-to-analog spectrum, dramatically expanding the engineerable behaviors available to synthetic circuits.

In nature, all-or-nothing responses to environmental stimuli often rely on cooperative self-assembly. “Several proteins called transcription factors self-assemble into a larger complex,” explained Caleb Bashor, PhD, assistant professor of bioengineeering and bioscience at Rice University and one of the co-lead authors of the current study. “Only when they come together is the switch thrown.”

Cooperative self-assembly was emulated in the current study by a modular system of synthetic protein components. In this system, the components can assemble into complexes of varying size. When engineered into cells, this system allows the programming of cellular responses to defined stimuli. For example, in the current study, yeast cells were programmed to respond to two different drugs that were administered in varying concentrations via a microfluidic device.

“Using a model-guided approach, we show that specifying strength and number of assembly subunits enables predictive tuning between linear and nonlinear regulatory response for single- and multi-input circuits,” the article’s authors wrote. “We demonstrate that assemblies can be adjusted to control circuit dynamics. We harness this capability to engineer circuits that perform dynamic filtering, enabling frequency-dependent decoding in cell populations.”

The concentration of component molecules produced inside the yeast rose and fell in response to the analog input—the concentration of drugs in the test chamber.

“Basically, these components bind to one another with extremely weak interactions,” Bashor said. “But all of those weak interactions add up, in a bigger complex, to something that’s really tight. So, when there’s very few of them floating around, they won’t form the complex. And when they reach a critical concentration, they see each other, and they can basically come together and form the complex.”

The sharpness of a response—one that happens quickly at precisely the intended time—is key for digital precision. “Engineering this type of response into transcription factors was central for allowing us to program cells to perform a diverse array of complex functions, such as Boolean logic, time-dependent filtering, and even frequency decoding,” said Ahmad “Mo” Khalil, PhD, assistant professor at Boston University and the corresponding author on the current study.

Bashor, Khalil, and colleagues designed activation complexes that contained as few as two transcription-factor components and as many as six, and their experiments showed that the larger the complex, the sharper the critical response.

Synthetic biologists designed a system for engineering cells that respond digitally to analog signals. Cells are preprogrammed to produce transcriptional protein components (top left) in proportion to the strength of an incoming signal. Upon reaching a critical concentration, components self-assemble (top right) into a protein complex that initiates transcription of a pre-programmed target gene. The more modules a self-assembled transcription complex contains (bottom, left to right), the more sharply it responds to the critical threshold. [Rice University]

“This work is a tour de force of synthetic biology that addresses a major question in how cells process information at the DNA level,” said Tom Ellis, reader in synthetic genome engineering in the department of bioengineering at Imperial College London, who was not involved in the study. “It’s well known that nature has perfected very powerful information processing with only a small number of parts, but deconvoluting precisely how this works is virtually impossible in human cells due to their complexity.

“By recreating the way human cells process information at the DNA level, but in a simple yeast cell model with synthetic parts, they have been able to recreate complex signaling from first principles. This is an excellent example of how thinking like an engineer can unlock a new way to answer major biology questions.”

Going forward, Bashor’s Rice lab plans to use the analog-to-digital converter and other synthetic gene circuits to explore and manipulate the regulatory programs that guide immune and stem cell functions with an eye on developing transformational cell-based therapeutics from engineered human cells.

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