Cells are one of nature’s greatest feats of engineering. They contain within the instructions for the entire lifecycle of our bodies. They are a critical building block for all life on Earth. But they also have their limitations.
Cells must be alive to produce proteins, one of the primary areas of interest in synthetic biology research. This presents a number of challenges. Lab-grown proteins have traditionally been created in vivo using expression systems based on mammalian cells, yeast and E. coli. This approach employs plasmids to express the desired proteins. But the process of transformation–introducing specific genes into the cell for the plasmids to encode–is very time-consuming. And even after the intended proteins have been produced, they can still be trapped in the cell. The cells must be lysed, typically requiring chemical, enzymatic or mechanical processes, so the proteins can be purified. All in all, protein expression through plasmids is extremely laborious. Moreover, it does not lend itself well to high-throughput protein expression and screening, an essential component for any production platform to meet the demands of modern synthetic biology.
And, then of course, there’s the fact that cells die. Cells in the lab need to be kept happy in an approximation of their ideal living conditions. Researchers must also be sure not to direct protein expression that would inadvertently kill the host E. coli cell. To generate certain, more toxic proteins, an entire precursor phase must be added– costing time, money and no small amount of frustration. Some projects are simply not possible because there is no way to circumvent the protein’s toxicity with a precursor phase.
So, as vital as living cells are to, well, life, their limitations create a bottleneck on the progress of 21st century biology. Wouldn’t it just be easier to do away with the “live” part of the cell and keep the rest?
Breaking out of the cell
Cell-free protein expression does just that. The technology retains the component mechanisms of a cell but leaves the hassle of cell cultivation and preservation behind. This opens up a world of possibilities. Now, proteins of interest can be generated quickly, consistently, and reliably. The viability of these proteins can also be tested at the same rapid pace.
Dr. Michael Jewett, at Northwestern University, spoke with SynBioBeta about the advances in high-yield cell-free gene expression. “These advances provide exciting opportunities to profoundly transform synthetic biology through new approaches to model-driven design[s] of genetic circuits, fast and portable sensing of compounds and next-generation education kits.”
Dr. Jewett also sees the cell-free benefits for “on demand bio-manufacturing of vaccines and therapeutics.” This is particularly important for developing new antibiotics, especially as instances of antibiotic resistance are increasing worldwide.
The ease with which companies like Arbor Biosciences are making cell-free technology accessible to any researcher has made them a leader in both R&D and the synthetic biology market. Their cell-free protein expression platform, myTXTL®, uses a master mix to create an open-reaction environment, free from the confines of cell walls or membranes. The master mix contains all the metabolic functions of E. coli, including the transcription (TX), translation(TL), and protein folding mechanisms, as well as amino acids and ATP regeneration systems. But it is not, in fact, a living organism. With the master mix in hand, “All that is needed to express your protein of interest is a DNA template, either plasmid or linear DNA, a tube, and a pipette,” says Dr. Evelyn Eggestein, a myTXTL Product Development Scientist. “It’s easily accessible and to control.”
“User-friendly” is a key factor in the continued evolution of synthetic biology. The days when only the most specialized experts could work in the field are rapidly fading. Contributing SynBioBeta writer Ian Haydon compares synthetic biology today to where computing was 50 years ago: experts only. But easy-to-use platforms like myTXTL are the next step in advancing and simplifying biotechnology. And this especially becomes obvious considering that the myTXTL platform also works in combination with linear DNA fragments that nowadays can be ordered cheaply and sequence-perfect from several providers such as IDT and Twist to screen millions of DNA samples without cloning into a plasmid vector. This type of screening has applications in basic research as well as novel diagnostic tools.
Multiple companies, as well as several iGEM teams supported by Arbor Biosciences, are developing paper-based assays with the form factor of a home pregnancy test. These card-like tests contained dried-down master mix, biosensors, and a fluorescent reporter molecule. All that is required to complete the test is the biological sample itself. These easily portable, pocket-sized tests have the potential to swiftly identify, help slow, or even contain disease outbreaks. They could also aid in diagnosis in the field, especially if refrigeration is unavailable, or be used to screen for genetic conditions in developing areas with poor laboratory coverage.
The future of cell-free
The potential of the paper-based form factor isn’t only bound to Earth. Freeze-dried master mix could be taken to Mars, moistened with water and voilà! protein production goes to Mars. This far-reaching mindset is in line with growing recognition for applications of synthetic biology in space. As always, those in the world of synthetic biology are looking far into the future.
What does this future look like for cell-free technology?
Now that Arbor Biosciences has successfully developed a platform suitable for high-throughput screening, its Marketing Director Matthew Hymes envisions developing cell-free systems with expanded novel capabilities and features. “E. coli has only so much basic machinery,” explains Hymes. “So can we add other elements to a cell-free system which drive specific protein folding or add translational modifications?” The answer is: yes we can! Traditional (in vivo) protein production platforms have already found solutions for how to produce proteins that typically don’t fold in the bacterial cytoplasm or how to site-specifically label a protein with small molecules for therapeutic and diagnostic use . “Now it’s time to adapt and integrate those approaches into cell-free systems,” Dr. Eggenstein says.
Dr. Jewett also emphasizes that cell-free systems “are the next stage in the evolution of metabolic engineering.” “[These systems] offer exciting opportunities to debug and optimize biosynthetic pathways before implementation in live cells and scale-up.” They can also carry out “design-build-test” iterations without the need to re-engineer organisms. They can even “perform molecular transformations when bio-conversion yields, productivities, or cellular toxicity limit commercial feasibility.”
Hymes acknowledges that cell-free technology isn’t currently up to handling the complexities of complete metabolic engineering. But when he speaks of cell-free metabolic engineering, it is in the tone of “when,” not “if.” The exact nature of science beyond the current cutting edge is unknown. But what is certain is that the future is arriving in leaps and bounds. The team at Arbor Biosciences is ready for it with open arms.0