Science: Successful Construction of Protein-protein Interaction-based Switches

Synthetic biology provides a way to engineer cells to perform new functions, such as emitting fluorescence when they detect a certain chemical. Usually, it is achieved by changing the cell's expression of genes that can be triggered by a certain input. However, because it takes time for cells to transcribe and translate essential genes, there is often a lag period between detecting events such as molecules and the resulting output.

Nowadays, in a new study, researchers from the Massachusetts Institute of Technology have developed an alternative method for designing such synthetic circuits, which relies entirely on rapid and reversible protein-protein interactions. This means that there is no need to wait for genes to be transcribed or translated into proteins, so synthetic circuits can be turned on faster, within seconds. The findings were published in the July 2, 2021 issue of Science, entitled "An engineered protein-phosphorylation toggle network with implications for endogenous network discovery". The corresponding author of the paper is Ron Weiss, a professor of bioengineering at the Massachusetts Institute of Technology.

Deepak Mishra, lead author of the paper and associate researcher in the Department of Bioengineering at MIT, said, "We now have a way to design protein interactions that occur at very fast time scales, and no one has been able to systematically develop this method before. We are reaching the point where any function can be designed on a timescale of a few seconds or less."

According to these authors, such synthetic circuits may be used to fabricate environmental sensors or diagnostic systems to reveal disease states or upcoming events, such as heart attacks.

Protein interaction

Within living cells, protein-protein interactions are an important step in many signaling pathways, including those involved in immune cell activation and response to hormones or other signals. Many of these interactions involve the activation or inactivation of another protein by the addition or removal of chemical groups called phosphates.

In this new study, these researchers used yeast cells to host their synthetic circuits and created a network of 14 proteins from species including yeast, bacteria, plants, and humans. They modified these proteins so that they could regulate each other in the network to generate signals in response to specific events.

Their network is the first synthetic circuit composed entirely of phosphorylated/dephosphorylated protein-protein interactions and is designed as a switching switch—a circuit that can quickly and reversibly switch between two stable states so that it can "remember" a specific event, such as contact with a chemical. In this case, the target is sorbitol, a sugar alcohol found in many fruits.

Once sorbitol is detected, cells store memories about this exposure in the form of fluorescent proteins localized in the nucleus. This memory will also be transmitted to future cell offspring.

These circuit networks can also be programmed to perform other functions in response to inputs. To demonstrate this, these authors also designed a circuit, which turned off the ability of cells to divide after detection of sorbitol.

By using large arrays of these cells, they can construct ultrasensitive sensors that respond to target molecular concentrations as low as one-billionth of a billion. Given rapid protein-protein interactions, signals can be triggered in just one second. With conventional synthetic circuits, it may take several hours or even days to see the output. "This extremely fast conversion will be important for the development of synthetic biology and expanding the types of possible applications," Weiss says.

Complex network

The switching network designed by these researchers in this study is more complex than most of the previously designed synthetic circuits. Once they established it, they wondered whether any similar networks might exist in living cells. Using a computational model they designed, they found six naturally occurring, complex switching networks in yeast that had never been seen before.

"We don't want to look for these switching networks in the past because they are not intuitive," Weiss says. They are not necessarily optimal or elegant, but we do find multiple examples of such switching behavior. It is a new, engineering inspired method for discovering regulatory networks in biological systems. "

These authors now want to use their protein-based circuits to develop sensors that can be used to detect environmental contaminants. Another potential application is the deployment of customized protein networks within mammalian cells as diagnostic sensors for the human body to detect abnormal hormone or blood glucose levels. In the long run, Weiss envisioned the designed circuit to enter human cells by programming to report overdose or an impending heart attack.

"In this case, the cell reports the information to an electronic device that alerts the patient or doctor, and this electronic device also has a chemical reservoir that can counteract the impact on the system," he said.

Collected by Creative BioMart, a recombinant protein provider.