Plasmids are widely used in basic and applied biology. These small round DNA molecules are used by scientists to introduce new genes into the target organism. Well known for their use in the production of therapeutic proteins such as insulin, plasmids are widely used in the large-scale production of many organic products.
However, the design and construction of plasmids remains one of the most time-consuming and time-consuming steps in biological research.
To address this problem, Behnam Engiad, Pu Xue, and other Urbana-Champaign researchers from the University of Illinois at the Center for Advanced Innovation in Bioenergy and Bioproducts (CABBI) have developed a universal and automated platform for designing and constructing a plasmid called Plasm. Their work was recently published in The nature of communication.
Plasmid creation begins with design. To help with this design process, PlasmidMaker has a user-friendly web interface through which researchers can intuitively visualize and assemble the perfect plasmid for their needs.
Once the plasmid has been developed, it is transferred to the PlasmidMaker team, and the order for the plasmid is placed at the Illinois Biological Foundry Advanced Biomanufacturing (iBioFAB), where the plasmid will be built. The iBioFAB, located at the Carl R. Vöse Institute of Genomic Biology (IGB) on Campus U of I, is a fully integrated computing and physical infrastructure that supports rapid fabrication, quality control and analysis of genetic constructs. It has a central robotic arm that carries laboratory equipment between devices that perform various operations such as pipetting, incubation or a thermal cycle.
The plasmid generation process is automated: samples are prepared by polymerase chain reaction (PCR) and purification, the DNA sequence is collected and transformed, and the plasmids are confirmed and frozen, all with little human involvement.
In addition to the automation and accuracy provided by iBioFAB, the PlasmidMaker platform also introduces a new very flexible method of assembling multiple DNA fragments into a plasmid using Pyrococcus furiosus organist (PfAgo) based on artificial restriction enzymes (ARE).
Restriction enzymes have long been used in the construction of plasmids, as they can cleave DNA molecules on certain sequences of bases, called recognition sequences. However, these recognition sequences are usually short, making it difficult to work with them. The short sequence is likely to occur several times in the DNA molecule, and in this case the restriction enzyme will make too many cuts.
“In previous DNA assembly methods, it was often difficult to find the right restriction enzymes that could cleave a plasmid and replace DNA fragments,” said Huimin Zhao, co-author and chair of Chemical and Biomolecular Engineering Stephen L. Miller (ChBE) in Illinois. « PfARE, based on earlier, provide greater flexibility and accuracy, as they can be programmed to search for longer recognition sequences on virtually any site. ”
With all the improvements it brings to the table, members of the CABBI team, one of four U.S. Bioenergy Research Centers funded by the U.S. Department of Energy, hope PlasmidMaker will accelerate the development of synthetic biology for biotechnology applications.
“This tool will be available to CABBI researchers, and we want to eventually make it available to all researchers at three other bioenergy research centers,” Zhao said. “If all goes well, we hope to make it available to all researchers around the world.”
Other co-authors of the manuscript are Nilmani Singh, CABBI Automation Engineer; Aashutosh Girish Boob and Chengyou Shi, CABBI ChBE graduate students; Vasily Andrey Petrov, CABBI software engineer; Roy Liu, CABBI student in computer engineering; Siddhartha Surianarayana Perry, undergraduate student at CABBI ChBE; Stephen Thomas Lane, CABBI iBioFAB Manager; and Emily Daniel Geiter, a former CABBI iBioFAB technician.