The team develops an automated platform for the production of plasmids

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The Illinois Biological Foundry for Advanced Biomanufacturing (iBioFAB) supports automated construction of plasmids through a new platform called PlasmidMaker. 1 credit

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

However, the design and construction of plasmids remains one of the longest and most laborious steps in biological research.

To solve this problem, Behnam Enghiad, Pu Xue and other researchers at the University of Illinois at Urbana-Champaign from the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) have developed a versatile and automated platform for designing and the construction of plasmids called PlasmidMaker. Their work was recently published in Nature Communication.

Plasmid creation begins with design. To facilitate this design process, PlasmidMaker features a user-friendly web interface with which researchers can intuitively visualize and assemble the perfect plasmid for their needs.

Once the plasmid is designed, it is submitted to the PlasmidMaker team and an order for the plasmid is placed with the Illinois Biological Foundry for Advanced Biomanufacturing (iBioFAB), where the plasmid will be constructed. iBioFAB, located at the Carl R. Woese Institute for Genomic Biology (IGB) on the U of I campus, is a fully integrated computational and physical infrastructure that supports rapid manufacturing, quality control, and l analysis of genetic constructs. It features a central robotic arm that transfers labware between instruments that perform separate operations such as pipetting, incubation or thermocycling.

An overview of PlasmidMaker. Credit: Enghiad et al.

The plasmid construction process is automated: samples are prepared by polymerase chain reaction (PCR) and purification, DNA sequence is assembled and transformed, and plasmids are confirmed and frozen, all with little involvement human.

In addition to the automation and precision offered by iBioFAB, the PlasmidMaker platform also pioneers a new, highly flexible method for assembling multiple DNA fragments into a plasmid using artificial restriction enzymes (AREs ) based on Pyrococcus furiosus Argonaute (PfAgo).

Restriction enzymes have long been used in the construction of plasmids because they can cleave DNA molecules at specific sequences of bases, called recognition sequences. However, these recognition sequences are generally short, which makes them difficult 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 was often difficult to find the right restriction enzymes capable of cutting the plasmid and replacing DNA fragments,” said Huimin Zhao, co-author and holder of the Steven L. Miller Professorship in Chemical and Biomolecular Engineering (ChBE) at Illinois. “PfAgo-based AREs offer greater flexibility and accuracy because they can be programmed to search for longer recognition sequences at virtually any site.”

With all the improvements it brings to the table, team members at CABBI, one of four US Department of Energy-funded bioenergy research centers in the United States, hope that PlasmidMaker will accelerate the development of synthetic biology for biotechnology applications.

“This tool will be available to CABBI researchers, and eventually we want to make it available to all researchers from the other three bioenergy research centers,” Zhao said. “If all goes well, we hope to make it available to all researchers around the world.”


New method of genetic engineering indispensable tool in biotechnological applications


More information:

Behnam Enghiad et al, PlasmidMaker is a versatile, automated and high-throughput end-to-end platform for constructing plasmids, Nature Communication (2022). DOI: 10.1038/s41467-022-30355-y

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University of Illinois at Urbana-Champaign


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