Engineers create protein circuits which respond in seconds

The growing field of synthetic biology allows engineers to create cells which perform novel functions, such as by altering cells to express genes that can be triggered by a specific input. However, a drawback is the long delay between an input (such as detecting a molecule of interest) and the resulting output, due to the time required for cells to transcribe and translate the necessary genes.

Now, synthetic biologists at MIT’s Department of Biological Engineering have developed an alternative approach to designing such circuits, which uses fast, reversible protein-protein interactions. This removes the need to wait while genes are transcribed or translated into proteins, cutting working times to seconds.

“We now have a methodology for designing protein interactions that occur at a very fast timescale, which no-one has been able to develop systematically. We’re getting to the point of being able to engineer any function at timescales of a few seconds or less,” said Deepak Mishra, lead author of the Science study.

Protein-protein interactions are critical steps in many signalling pathways, including those involved in immune cell activation and hormone response. These interactions often involve one protein activating or deactivating another by adding or removing chemical groups called phosphates.

In this study, the researchers used yeast cells to host their circuit and created a network of 14 proteins from a huge range of species, including yeast, bacteria, plants, and humans. The researchers modified the proteins such that they could regulate each other in the network, outputting response to a particular event. The network is a 'toggle switch': a circuit which can quickly and reversibly switch between two stable states, allowing it to maintain a 'memory' of an event; in this case, the presence of sorbitol.

Once sorbitol is detected, the cell stores a memory of the exposure in the form of a fluorescent protein localised in the nucleus. This is passed on to future generations of cells. However, the circuit can be reset when exposed to a different molecule, in this case isopentenyl adenine.

The MIT researchers’ network is the first synthetic circuit to consist entirely of phosphorylation/dephosphorylation protein-protein interactions. Networks of this sort can be programmed to perform a range of other functions in response to an input; they demonstrated their versatility by designing a second circuit that blocks cell division after detecting sorbitol.

According to the researchers, large arrays of these cells could be used to create ultrasensitive sensors which respond to tiny traces of the target molecule (as low as parts per billion). The speed of protein-protein interactions means that the signal could appear in as little as a second, compared with hours or even days for conventional synthetic circuits.

“That switch to extremely fast speeds is going to be really important moving forward in synthetic biology and expanding the type of applications that are possible,” said Professor Ron Weiss, an expert in electrical engineering and computer science.

The network designed by Weiss and his colleagues for this study is larger and more complex than previous synthetic circuits. The researchers – interested in seeing whether comparable networks occur naturally – used a computational model to identify six naturally occurring 'toggle networks' in yeast which had never been seen before.

“We wouldn’t think to look for those because they’re not intuitive. They’re not necessarily optimal or elegant, but we did find multiple examples of such toggle switch behaviours,” Weiss continued. “This is a new, engineered-inspired approach to discovering regulatory networks in biological systems.”

Protein-based circuits like these the researchers created could have applications in sensors in the environment and medicine, potentially revealing disease states or – when 'programmed' into human cells – giving warnings of imminent heart attacks or drug overdoses, and respond by releasing a reservoir of chemicals to counteract the event. They hope to use protein-based circuits to create sensors for environmental pollutants.