Cars have cruise control. Jet planes have auto pilot. Factories have industrial process control. And now, thanks to synthetic biology, cells have human-engineered integral feedback control. In a proof-of-concept study, Escherichia coli bacteria have been equipped with a controller in the form of a cybergenetic regulatory network, that is, a set of interacting biological circuit elements.
These elements accomplish for bacterial cells what integral feedback loops have long accomplished in industrial plants—which is fitting, since bacterial cells and other types of cells are used as tiny chemical factories in biotechnology applications.
Just as integral feedback loops sustain optimal productivity on the factory floor, such feedback loops could ensure that producer cells release desirable products—vitamins, medications, chemicals, and biofuels—at high, stable rates. Integral feedback loops could also result in designer cells that could maintain optimal levels of chemicals inside a patient’s body, helping resolve conditions such as diabetes or thyroid deficiency. Finally, they could improve cancer immunotherapy, ensuring that immune cells are active enough to fight tumors but restrained enough to avoid attacking healthy tissue.
The basic idea is to introduce a mechanism that can fine-tune cellular activity and thereby achieve useful ends—useful so far as bioengineers and translational scientists are concerned. Integral feedback loops that evolved naturally are already hardwired into cells. For example, integral feedback loops maintain constant concentrations of substances in the blood.
The presence of natural integral feedback loops was established several years ago by scientists based at ETH Zurich. “These kinds of integral controllers are extremely resistant to unexpected environmental disturbances,” said Mustafa Khammash, a professor at ETH Zurich’s department of biosystems science and engineering. This probably explains, he continued, why the integral feedback principle, which is ubiquitous in technology, has prevailed in evolution.
Khammash and colleagues applied what they learned from nature to genetically engineer a synthetic controller. In an article (“A universal biomolecular integral feedback controller for robust perfect adaptation”) that appeared June 19 in Nature, the scientists reported that they installed their controller in living cells. The controller, they emphasized, is tunable and adaptive.
“[Our] rationally designed integral controller … represents a proof-of-concept design that establishes the feasibility of engineering robust homeostasis in synthetic biology,” the article’s authors wrote. “A suitably optimized version of this controller with expanded dynamic range should find wide applications in all scenarios in which protein expression must remain tightly regulated at the desired level, independent of other intervening processes.”
The authors noted that the synthetic realization of integral feedback in living cells has remained elusive owing to the complexity of the required biological computations. Nonetheless, the authors developed a mathematical proof that there is a “single fundamental biomolecular controller topology that realizes integral feedback and achieves robust perfect adaptation in arbitrary intracellular networks with noisy dynamics.”
Khammash and his interdisciplinary team of control theorists, mathematicians, and experimental biologists explained that their feedback mechanism relies on two molecules—call them A and B—that bind to each other to become inactive. Together, these two molecules can maintain a constant concentration of a third molecule, C. The system is designed so that molecule B promotes the production of C, while the production rate of A depends on the concentration of C. The feedback loop consists in the fact that when C is abundant, more A will be produced, which will inactivate more B, which in turn will cause production of C to fall.
The ETH scientists used their mechanism to control the production of a green fluorescent protein in Escherichia coli bacteria. Thanks to the feedback controller, the bacteria produced a constant amount of the fluorescent protein—even when the scientists, who wanted to test the system, attempted to suppress its production using strong inhibitors. In a second experiment, the researchers managed to produce a bacterial population that grew at a constant rate in spite of the scientists’ attempts to disrupt growth, again in an effort to test the feedback mechanism.
“This adaptation property is guaranteed both for the population-average and for the time-average of single cells,” the scientists pointed out. “Our results provide conceptual and practical tools in the area of cybergenetics for engineering synthetic controllers that steer the dynamics of living systems.”
Khammash and colleagues succeeded in building an integral controller completely from scratch and testing it within a living cell partly as a result of their earlier work on calcium homeostasis. According to Khammash, studying how calcium concentration in the blood is regulated is a good way to learn how integral controllers work in biology.
The concentration of calcium is tightly regulated at a value of approximately 95 milligrams per liter of blood, regardless of how much calcium a person ingests in food. This rate even remains constant during lactation when lots of calcium is drawn from the blood in order to produce milk. “A constant level of calcium is essential to the proper functioning of many physiological processes, including muscle and nerve function or blood clotting,” Khammash said.
The hormone PTH works as one of two feedback agents in the body in this context: PTH promotes the mobilization of calcium from bone tissue into the bloodstream. The lower the concentration of calcium in the blood, the more PTH is produced by the parathyroid glands. “This is one part of the body’s response when the levels of calcium are too low,” Khammash noted.
But to bring the concentration of calcium completely back to normal after a sudden spike or drop, he added, a second mechanism is required. This role falls to a biologically active form of vitamin D3, which promotes the absorption into the bloodstream of calcium from partially digested food in the small intestine. However, production of this active form of vitamin D3 in the kidneys is dependent on the concentration of PTH.
Together, these two hormones are responsible for ensuring that the calcium concentration in the blood over time strays as little as possible and for as short a time as possible from its normal level—or, in other words, that the “integral of deviation with respect to time,” as a mathematician would put it, approaches a constant. Therefore, such a control mechanism is called integral.