Synthetically engineered protein circuits

Eureka WSRC
Jan 18, 2022
5 min read

“The activity characteristic of professional engineering is the design of structures, machines, circuits, or processes, or of combinations of these elements into systems or plants and the analysis and prediction of their performance and costs under specified working conditions.”

- Morrough Parker O’Brien (1954)

The trillions of cells within our bodies form an intricately interconnected network that

works much in the same way as a computer. Just as a computer has software that contains instructions on how to respond to the inputs received, cells contain genetic code or DNA that instruct the proteins in the cells to respond to the signals around it telling them what to do. DNA or genetic code also controls instructions on building proteins that are an essential part of the fabric and function of the human body. So an error in the genetic code can cause severe issues to the instructions it gives out, disrupting the functioning of the cells.

Errors in the genetic codes can also vary greatly. Sometimes a part or whole of a gene is defective or missing, at other times a gene may change or mutate in adult life leading to the production of abnormal proteins causing the cells to behave differently than they are expected to. Any of these variations contribute to health problems or diseases including autoimmune disease as it can disrupt how proteins respond to the signal received. A good example of such a mutation is cancer, where the cells continue to divide and multiply even when there are signals around them to turn off and stop multiplying after the first division.

This has long been understood and to solve the issue, scientists have been developing methods like gene therapy where they work on different ways to correct or replace the problem causing genes. If genes are absent or missing, they can be added to help the body fight or treat disease, damaged genes can be replaced with healthy ones or the genetic code of the problem causing genes can be corrected by inserting the right set of code at the exact same place. But it is incredibly challenging and difficult to locate and replace the gene or rewrite the code in the exact same spot.

So researchers are now working on a slightly different approach to solving the problem using synthetic biology, an engineering discipline that uses mathematical modelling of cellular biology to create, control and programme cellular behaviour. Using synthetic biology, researchers have experimented on adding a switch known as a toggle switch that can be used to simply turn off the damaged cells instead of attempting to repair or replace the damaged genetic code. To be able to do this, they engineered synthetic protein circuits, similar to natural protein circuits.

Understanding protein circuits

Every cell consists of thousands of different proteins creating a circuit or network of interacting proteins. One of them (the receptor) can detect an external signal causing a change in behaviour and pass the signal on to the next one and the next, creating a domino effect making the cell do different things. This network of interacting proteins is a protein circuit. There are many different kinds of protein circuits and each one can generate different cellular programs creating different reactions.

The working of engineered protein circuits

Engineered synthetic biological regulatory circuits can be added to human cells and a broad variety of circuit-level functions can be induced in the cells to control the behaviour of the protein and thus of the cell. The first-ever "genetic toggle switch," designed to control the activity of genes, was engineered in the year 2000 by scientists at Boston University. They worked with the bacteria Escherichia Coli, and were able to successfully switch the expression of genes between stable on and off states. A wide range of computational circuits for cells have been developed since enabling the creation of programmable cells with decision-making capabilities for a variety of applications. In this system, termed CHOMP (circuits of hacked orthogonal modular proteases), circuits can perform complex functions and offer a platform to facilitate protein circuit

engineering for biotechnological applications.

The experiment

The researchers designed and created an engineered protein level circuit consisting of a sensor and an effector/terminator protein to show how they can be assembled in a very easy fashion to give many types of logical functions to the gene expression. This was shown in the experiments conducted by MIT researcher Dr Timothy Lu in 2017, and Caltech university researchers in 2018.

The sensor was designed to detect a specific enzyme present in the cancer cells that triggers the uncontrolled growth circuit of cells. The promoter was designed to turn on only when it detects this enzyme. The synthetically engineered effector protein or terminator protein is similar to a natural protein that can turn off a protein circuit. The researchers attached the sensor to the effector protein creating a circuit that could be used to turn off the protein circuit and thus stop the cell from responding to the enzyme in undesired ways. In short, the sensor would detect the enzyme and turn on, thus activating the effector protein to shut down the circuit. To detect the enzyme, the sensor and effector protein would have to be part of the natural protein circuit of a cell so researchers developed ways to insert the engineered DNA code (sensor-effector protein code) into a cell. They used different ways to do it like loading it into circular strings of DNA code called plasmids and coating them with lipids so that the cell would absorb the code, considering it to be natural DNA code. This method termed ‘transfection’ would also eliminate the need of editing the cell's own DNA.

The protein circuits developed in the first step were effective in killing cancer cells however they also presented an issue - the core circuit killed healthy cells too as it shut down the whole system even if a small amount of signal was detected. Therefore if we want to use synthetic biology to cure cancer, only the cancer cells need to be shut down and not the healthy cells. Researchers at Caltech University further demonstrated an experiment on cancer cells in a lab setting. They developed a threshold filter that could detect a certain amount of enzymes in the cancer’s grow and divide circuit and when the threshold was hit by the enzyme, the effector would be activated to turn off the circuit, and ensure that it stays off. This made sure the effector turned on the shutdown program only if a cell was overusing its grow-and-divide circuit, so the non-cancerous cells were protected and the healthy cells were left alone. This helped change the fate of a cell at the flick of a

chemical switch. The aim of this technology is to develop therapeutic "circuits" that would be highly

targeted; instead of affecting all cells indiscriminately, the therapeutic circuits could detect when something goes wrong on the cellular level and fix it accordingly. They can thus be administered to carry out a function, and then go away once their purpose has been served without making a permanent change to the natural DNA.

Conclusion

Until recently, writing DNA code into cells was just in science fiction movies. But today young scientists in universities around the world are learning to use synthetic biology, directly reprogramming cells to fix them instead of treating a diseased organism with drugs. Yet, individual cells grown in a petri dish are not the same as full living organisms. A lot more needs to be learnt about how to safely and effectively write these cellular software updates before they can be used in people. Even so, the protein circuit technique offers great promise for the future of human health.

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