Cornell University scientists have created an engineered version of the bacterium Vibrio natriegens, which they say will offer researchers a low-cost and scalable platform for carrying out synthetic biological experiments. The organism, they suggest, could compete economically with Escherichia coli—a bacteria that is commonly used as a research tool for synthesizing proteins.

The team reported that the engineered Vibrio natriegens behaves as an inexpensive multiplier—much like having a photocopier in a test tube—that can be engineered within hours. The organism is also naturally competent to plasmids—the small pieces of DNA that act as the instruction manuals for making the protein of interest—and can effectively transform plasmids, without costly incubators, shakers or deep freezers. The overall system, the researchers hope, will give labs a simple process for evaluating protein variants, whether for creating pharmaceuticals, synthetic fuels, or sustainable compounds against weeds or pests.

“The microbe is a radically simple solution to a hard problem,” said research lead Buz Barstow, PhD, assistant professor of biological and environmental engineering.  “It’s really easy to produce,” added David Specht, PhD, a postdoctoral researcher in the Barstow laboratory.

Specht is first author of the team’s published paper in PNAS Nexus, titled “Efficient Natural Plasmid Transformation of Vibrio natriegens Enables Zero-capital Molecular Biology,” in which the researchers concluded “… naturally competent V. natriegens could compete with E. coli as an excellent chassis for low-cost and highly scalable synthetic biology.”

Researchers studying proteins use plasmids to generate protein variants of interest. The plasmids are introduced into bacteria in which many copies are then produced. E. coli is commonly used as the platform for multiplying and manipulating plasmids for protein engineering. However, the process is expensive, as the bacteria may typically be purchased from manufacturers, must be kept cold, and then expensive equipment is needed to sustain the organisms. Modified E. coli may also be very fragile.

“Bacterial competent cells derived from E. coli are a critical component of modern molecular biology,” the authors explained. “While these are easy to produce, the process is tedious and requires the tools of a typical biology lab, limiting the democratization of synthetic biology.”

Over recent years scientists have turned their attention to the fast-growing microbe, Vibrio natriegens, as a potential next-generation E. coli replacement that could act as an alternative host for synthetic biology. In addition to its extremely fast growth rate, with an optimal doubling time observed to be less than ten minutes in rich media, V. natriegens has “a number of advantages as a host for biotechnology,” particularly for applications in metabolic engineering, the team continued.

The V. natriegens platform developed by Barstow and colleagues gives researchers the ability to transform plasmids using a simple process, by which the cells are made competent, transformed, and recovered in the same media, without concentration or media exchange. The process can all take place at room temperature, and with no need for specialized equipment, which makes the platform comparatively inexpensive. “Cells of our engineered strain are produced and transformed in this singular media, without any exchange, concentration, or separate media recovery, enabling the entire process to be able to be completed with no capital equipment at all, or enhanced with only the use of an incubator and deep freezer,” the investigators further commented.

The cells in addition retain their naturally competent state when frozen, and just need to be thawed. “The engineered naturally competent cells can be frozen and conveniently thawed for later use, to our knowledge for the first time by any researcher,” the team stated. The V. natriegens cells also grow quickly. According to the published results, a transformation started at 0900 will by 1700 yield visible single culturable colonies.

“As scientists, we don’t often know precisely what those regulatory or molecular sequences should be to achieve our goals,” said Barstow. “So, we must test a lot of variants, and Vibrio natriegens allows researchers to scale up that process of testing.”

The V. natriegens organism is not complicated, Specht added. “It’s so simple to make that someone with limited resources—like high school labs, home inventors or startup biological businesses—can do it.”

Co-author Timothy Sheppard, PhD, compared the simplicity of V. natriegens in conducting synthetic and molecular experiments to using a simple writing instrument hundreds of years old: “We’ve found nature’s pencil for cloning and conducting synthetic biology.”

Summarizing their developments and results, the authors concluded “… we demonstrate that this strain can be used for efficient plasmid transformation, using a simplified shared media for competence expression, incubation, and recovery, enabling simple, scalable, low-capital plasmid engineering using V. natriegens as a tool … This work will be of interest to a broad spectrum of researchers including the growing V. natriegens community, those interested in directed evolution and automation, and those that might lack traditional laboratory equipment.”

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