Engineering cellular function without living cells

A systematic method for studying and even predicting gene expression – without using cells.

Engineering cellular function without living cells
© Zoe Swank / 2019 EPFL

The proteins called transcription factors are responsible for the activation and deactivation of genes in living cells. Although, this mechanism is literally complex and scientists are spending years trying to reveal their secrets.

Until now, scientists have used specific DNA sequences into cells and observed how cells react. But, this method is complicated enough that it differs from experiment to experiment. By taking a step forward, scientists at EPFL’s Laboratory of Biological Network Characterization (LBNC) have developed a quantitative, replicable method for studying and even predicting gene expression.

They have developed a cell-free system in combination with a high-throughput microfluidic device to comprehensively study the different tuning mechanisms of a synthetic zinc-finger repressor library, whose affinity and cooperativity can be rationally engineered.

Nadanai Laohakunakorn, a co-author of the study, explains how the method works: “First we extract material from inside the cells. This ‘cell-free’ system consists of enzymes and chemicals that the cells use to carry out their normal biological processes. Interestingly, we can restart gene expression outside the cell by feeding the extract with fuel and information, in the form of high-energy phosphates and DNA. Because the process closely mimics what happens in living cells, we can use our platform to investigate a range of biological phenomena without having to modify living cells each time.”

During the study, scientists observed various cell-free reactions on a microfluidic chip. Testing in different cases, they build a quantitative library of synthetic transcription factors, which allowed us to predict the influence of a given protein on a gene.

Zoe Swank, another co-author of the study said, “Our method can be extended to build fairly complicated systems.”

The scientists’ method has several advantages:

  • First, cell-free systems can imitate systems within cells, yet they are much simpler, and their mechanisms can be modeled mathematically. This means that they can help contribute to understanding more complex biological phenomena by breaking them down into simpler pieces.
  • Second, cell-free systems are robust and remain stable after freezing (and even freeze-drying), which allows them to be produced on a large scale and deployed in applications from low-cost diagnostics to the on-demand production of biologics – such as vaccines – for personalized medicine.
  • And third, because they are not alive, cell-free systems can be used to produce compounds that go beyond the scope of traditional biomanufacturing methods. And they pose no risk of self-replication or biocontamination outside the laboratory environment.

During the study, scientists assembled a number of genes from their library to construct a biological logic gate. This biological logic gate takes an input of transcription factors and generates a binary output: the gene is either on (activated) or off (repressed).

Laohakunakorn said, “Numerous logic gates exist naturally within living cells, which use them to regulate normal biological function. By building artificial gates, we gain the ability to introduce new functions into cells for therapeutic purposes, for example. The cell-free system is the first step in this direction, and future work could involve optimizing the design of our transcription factors using the platform, before deploying them directly in a cell-free application, or reintroducing them back into living cells.”

The research has been published in PNAS.