Enzymes and other chemical reagents are crucial for all sorts of life sciences research. Typically, they’re produced in industrial processes by modified bacteria; then they’re extracted and purified. But scientists in many parts of the world struggle to access such commercial reagents, due to either high costs or difficulties importing them from abroad.
A recent study published in PLOS One offers an innovative solution: Researchers can simply dry and use the whole bacteria, with no need to separate and purify the reagent products. This greatly reduces costs, makes transport easier, and could allow scientists across the world to produce usable reagents in their own labs.
The simple process could be a game changer for research groups in developing countries. “These [reagents] are well-suited for resource-poor settings,” says coauthor Andrew Ellington, the synthetic biologist who led the research. “And I think in many ways a godsend for resource-poor settings.”
“Technologies or other techniques like these can really help countries like India to become independent to some extent,” notes Shivani Upadhyaya, a postdoc in plant and microbial biology at the University of California, Berkeley, who recently completed a PhD at the Tata Institute of Fundamental Research in Mumbai, India.
Upadhyaya, who was not involved in the new research, says projects in India frequently encounter delays because of shortages of reagents, which are usually shipped from Europe and the United States. Deliveries can take months.
To help to avoid such delays, Ellington and his colleagues at the University of Texas at Austin have worked for several years to develop reagents that can be used without purification. The process is not only cheaper, but also eliminates the need to keep the reagents at very low temperatures, which expands and simplifies the ways they can be stored and shipped. In 2018, the team showed that Escherichia coli bacteria used to express a range of useful proteins, including enzymes used to amplify DNA in the commonly used polymerase chain reaction (PCR), could be freeze-dried and then used as “cellular reagents” in lab procedures.
In the new study, the same team devised a way to avoid freeze-drying the cells. Instead, they show the bacteria can be grown and dried in a standard lab incubator with simple desiccating chemicals. That should put the procedure within reach of most labs across the world, they say.
“As long as the cells can be made permeable or broken in the context of the reaction itself, then the other components of the cell, all of the dirt that we usually clean away from an enzyme, don’t seem to matter a whole lot,” says Ellington.
The initial step is the same as the commercial production of such reagents. The scientists used plasmids to introduce the genes necessary to express a protein into E.Coli and grew the bacteria in a liquid culture for 24 hours. Spun down in a centrifuge, the protein-expressing bacteria were harvested, resuspended in small tubes, and sealed into jars that contained calcium sulfate desiccated pellets. After 1–2 days, all the liquid had evaporated and the tubes of now-dry bacteria were sealed.
Stored at room temperature for months, the bacteria could then be rehydrated and used as reagents in various reactions. “You could do PCR. You could even do Gibson cloning [a method that joins multiple DNA fragments],” Ellington says. This reduces the costs of reagents 10–100 fold, he adds.
Just as important, it allows for convenient transport. Writing in the new paper, the group describes how they sent dried cells by FedEx courier and then in personal luggage to collaborators in Cameroon and Ghana, where they were successfully used in lab reactions.
Julius Lucks, a synthetic biologist at Northwestern University in Evanston, Illinois, says the latest study is the most powerful demonstration so far of what those in the field call “cell-free” biology, a growing research area that aims to harness the reactions and molecular machinery that operate inside cells without the need for living cells. Previous work still required access to sophisticated equipment, Lucks says.
“I think the major innovation of this paper is really focusing on that bottleneck,” Lucks says, “trying to make it easier for people to make these reactions and explore how these reactions can be impactful for their own needs.” An early real-world use could be in disease diagnostics, he adds. Early in the COVID-19 pandemic, for example, lab work was held back by a lack of reagents. (See Inner Workings: Molecular biologists offer “wartime service” in the effort to test for COVID-19.)
For cell-free biology to prove its worth, scientists now need to make sure the results are as reliable as using purified enzymes and that batches prepared in different labs with little quality control perform the same. “They’ve really shown that this is possible, but now let’s have a hundred different labs make them and test them and see if it really is robust,” Lucks adds. “And if it’s not, if there is variability, let’s figure out how to control that.”
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