Combating COVID-19 with Cell-Free Expression

Going Cell-Free

Rewiring a living system is tricky. Cells are already biologically programmed to achieve their own functional goals, not the goals of the researcher. To circumvent this problem, synthetic biologists remove the cell wall and extract the cell’s molecular machinery, including the core factors needed for transcription and translation. This cell-free expression system can then be coerced to produce and even detect proteins of interest. 

“It's like if you took a car, you lift up the hood, pull out the engine, and you repurpose it for something else. We're repurposing the molecular machinery to do some objective function,” said Jewett. Working outside a cellular system prevents the cell from responding and changing its gene expression profile, ensuring a reproducible and stable molecular expression system.  

“The complexities itself are much lower than that of the living cells, and it's easier to control the different features in that you can dictate, at least at the outset, what [compounds] are there and how much is there,” said James Collins a professor of biological engineering at the Massachusetts Institute of Technology.

Scientists have used cell-free expression systems for two decades as a basic research tool to make discoveries about the living world. In fact, scientists used cell-free systems to uncover the genetic code in the 1960s. However, many of these early systems were small-scale, did not last very long, and could not make complex proteins. Within the last 20 years, researchers have addressed each of these problems, transforming cell-free expression systems from a basic tool into a useable technology platform.

“The systems now, instead of lasting five minutes, last 15 to 20 hours in batch reactions. They can make really complex proteins all the way up to full-length antibodies, which are used in medicine. They can carry out pretty complex integrated circuitry that can basically detect, sense, and respond to something just like a cell,” said Jewett. 

Researchers also developed better ways to support cell-free expression systems by supplying the system with glucose as a source of energy and other biological compounds such as amino acids needed to produce proteins. Scientists even solved the problem of scalability. “If you imagine running a PCR reaction, which is typically two or five microliters, in a 1000-liter scale, that's what we're doing economically, which is crazy. In fact, many people kind of deemed that impossible 10 years ago, but it's happening,” said Jewett. 

Cell-free expression systems can be rapidly pre-assembled and stored in a laboratory freezer, or freeze-dried in a powdered form. This eliminates the need for researchers to regrow cells. “The problems then reduce to automated liquid handling,” said Jewett. Automated liquid handling robots enable researchers to run more samples at a time for rapid library screening or analyzing genetic parts and gene circuits. 

Making cell-free expression systems is an art that, much like any other laboratory technique, takes skill, practice, and time. Some researchers make their own cell-free expression systems, but others purchase commercial ready-to-use master mixes. These mixes, such as the myTXTL® Cell-Free Expression System from Arbor Biosciences, come pre-loaded with all the molecular components for a given reaction so that the researcher only has to add their DNA sample for reliable and robust high-yield protein expression.

“The homebrew has some really nice efforts by Michael Jewett and his team to get them highly functional, but when we make our own, we're not as good and so it often doesn’t have the functionality level that we need,” said Collins. “My team has used Arbor Biosciences; they have very good products. In the cell-free world, they're one of the go-to [companies].” Like homebrewed systems, purchased premixed cell-free systems can also be paired with automated liquid handling for high-throughput and efficient library screening or analysis. 

Pivoting

These advances made it possible for synthetic biologists to develop practical technologies that mitigate disease and impact society. When the COVID-19 pandemic struck, synthetic biologists pivoted the cell-free expression technology that they were using for other diseases to develop new diagnostics, materials, and treatments specifically for COVID-19. 

Keith Pardee, a synthetic biologist at the University of Toronto was working on a three-year project to send Zika virus diagnostic testing kits to low- and middle-income countries. “When the COVID-19 outbreak happened, we thought this is obviously a natural thing for us to do. So, we basically are taking that platform for Zika and applying it to SARS-CoV-2,” said Pardee.

Pardee extracts enzymes needed for transcription and translation from Escherichia coli to create a cell-free expression system that senses parts of the SARS-CoV-2 genome and triggers a molecular switch to produce a reporter protein. It also eliminates the need to purify RNA from the sample and can replace RT-PCR for more rapid diagnostic testing. “Because we didn’t have that black box of working with a cell, we were able to get sensors very quickly, maybe within two days of having the synthetic DNA arrive in the lab, we had sensors working,” said Pardee. 

Pardee will send COVID-19 diagnostic kits in two waves to the same countries that he was planning to send the Zika kits, only now he has added Toronto, Canada to the list. The first wave will supply 1000 test kits a day for two weeks. The second wave will contain a “lab-in-a-box,” that could convert a common microbiology lab into a diagnostic testing lab to maintain a sustained testing capacity.

Similarly, James Collins was developing a suite of clothing-based sensors for healthcare workers, first responders, and military personnel to detect the presence of viruses or compounds. “The idea would be like a lab coat of the future,” said Collins. He conceived the idea during the Ebola outbreak of 2014. Collins and his team freeze-dried cell-free expression systems and locked them onto a piece of paper, and later into fabric. They then showed that the system could be rehydrated and transcription and translation activated by adding liquid.

“We were revising this publication when the pandemic hit, and realized that we could embody the same technology into facemasks by having an insert that can be added. If a person is infected, they'll give out particles in water vapor—that is coughing, sneezing, talking—and that itself could activate these freeze-dried components,” said Collins. If a person were infected, the mask would produce a fluorescent signal that could be detected using a handheld fluorometer. 

Collins’s previous discoveries in synthetic biology also contributed to the technology behind the Moderna vaccine for COVID-19, which is in the late-stages of development. Ten years ago, with George Daly and Derek Rossi, Collins developed a stable synthetic mRNA system to express proteins directly in cells. They used the technology to efficiently reprogram induced pluripotent stem cells, but mentioned in the paper that the technology could be used for RNA-based vaccine development. 

“I've been so motivated by so many scientists, by how much we're all redirecting; we're pivoting,” said Jewett. Four months ago, Jewett’s research focused primarily on developing water-based diagnostics for identifying toxins. Using the same cell-free expression approaches, Jewett is now developing CRISPR-based diagnostics for rapid COVID-19 detection and working on antiviral frontline approaches to stop COVID-19 infection. 

Using cell-free expression systems, Jewett’s team identifies proteins that could cloak the COVID-19 spike protein and prevent its connection to cell receptors. Identifying good clones using classic mammalian cell-culture can take 12 -18 months. “We need two months. We need four weeks. We need to have technologies that can meet the pace of this pandemic,” said Jewett. Instead of growing cells, Jewett and his team thaw pre-built, frozen cell-free expression stocks, add DNA, and identify good candidates within a day. 

If other laboratories are interested in pivoting but are unable to pre-assemble frozen stocks or lack the technical experience needed, they can reach out to commercial providers, such as Arbor Biosciences for reliable cell-free expression systems. Arbor Biosciences is adept at developing robust cell-free systems that can be immediately implemented in COVID-19 research or in other disease-based research for rapid, efficient, and dependable discovery. 

“In this era of emergent and reemerging pandemic outbreaks, what we need is speed. Cell-free systems really offer this exciting component, and the technology is well suited to address that need,” said Jewett. Cell-free expression systems alone will not solve the COVID-19 pandemic, but it can complement existing technologies. “Research is just developing. We, like many others, have been conceiving ideas and schemes and now's the time,” said Jewett. “We have to make an impact with whatever we can.”

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