Platform Automates Plasmid Design and Construction

Researchers in Illinois have developed an automated platform for plasmid production that they hope will streamline one of the most time-consuming and labor-intensive steps in biology research.

Plasmids are small, circular DNA molecules used by scientists to introduce new genes into a target organism. Well known for their applications in the production of therapeutic proteins like insulin, plasmids are broadly used in the large-scale production of many bioproducts and have extensive use in basic and applied biology.

However, designing and constructing plasmids remains one of the most time-consuming and labor-intensive steps in biology research. To address this, Behnam Enghiad, Pu Xue, and other University of Illinois Urbana-Champaign researchers at the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) have developed a versatile and automated platform for plasmid design and construction called PlasmidMaker. 

To aid in design, PlasmidMaker features a user-friendly web interface with which researchers can intuitively visualize and assemble the perfect plasmid for their needs. Once designed, the plasmid is submitted to the PlasmidMaker team, and an order for the plasmid is placed at the Illinois Biological Foundry for Advanced Biomanufacturing (iBioFAB). iBioFAB, located at the Carl R. Woese Institute for Genomic Biology (IGB) on the University of Illinois campus, is a fully integrated computational and physical infrastructure that supports rapid fabrication, quality control, and analysis of genetic constructs.

The plasmid build process is automated. Samples are prepared through polymerase chain reaction (PCR) and purification, the DNA sequence is assembled and transformed, and the plasmids are confirmed and frozen, all with little human involvement.

The PlasmidMaker platform also pioneers a new highly flexible method for assembling multiple DNA fragments into a plasmid using Pyrococcus furiosus Argonaute (PfAgo)-based artificial restriction enzymes (AREs). Restriction enzymes have long been used in plasmid construction, as they can cleave DNA molecules at specific sequences of bases, called recognition sequences. However, these recognition sequences are usually short, making them hard to work with. A short sequence is likely to occur multiple times in a DNA molecule, in which case the restriction enzyme would make too many cuts.

“In previous DNA assembly methods, it would often be hard to find the right restriction enzymes that can cut the plasmid and replace the DNA fragments,” says Huimin Zhao, co-author and the Steven L. Miller Chair of Chemical and Biomolecular Engineering (ChBE) at University of Illinois. “The PfAgo-based AREs offer greater flexibility and precision, as they can be programmed to seek out longer recognition sequences at virtually any site.”

With all the improvements it brings to the table, the team members at CABBI—one of just four U.S. Department of Energy-funded Bioenergy Research Centers across the United States—hope that PlasmidMaker will accelerate the development of synthetic biology for biotechnological applications.

“This tool will be available to CABBI researchers, and we want to eventually make it available to all researchers at the other three Bioenergy Research Centers,” Zhao said. “If things go well, we hope to make it available to all researchers everywhere.”

A proof-of-concept for PlasmidMaker is described in a recent issue of Nature Communications.