PlasmidMaker is a versatile, automated, and high throughput end-to-end platform for plasmid construction

University of Illinois Urbana-Champaign researchers, led by professor Huimin Zhao, developed a versatile and automated platform for plasmid design and construction called PlasmidMaker.
PlasmidMaker is a versatile, automated, and high throughput end-to-end platform for plasmid construction

Plasmids are used extensively in synthetic biology. However, the design and construction of plasmids, specifically the ones carrying complex genetic information, remains one of the most time-consuming, labor-intensive, and rate-limiting steps in performing sophisticated biological experiments. In recent years, biofoundries have shown great power in the automation of plasmid construction with increased throughput and accuracy1, 2. However, these platforms still lack flexibility in the construction of plasmids with virtually any DNA sequence. We aim to create a robust, versatile, and automated end-to-end platform for plasmid construction named PlasmidMaker that enables scarless construction of any plasmid in a high throughput manner.

To implement this platform, we developed a versatile, scarless, parallel, robust, and accurate method for assembly of multiple DNA fragments using Pyrococcus furiosus Argonaute (PfAgo) based artificial restriction enzymes (AREs)3. Then we designed both frontend and backend software for customers to build their specific DNA fragments using a user-friendly web interface and for our technicians to collect essential information that is required for DNA assembly, respectively. Finally, we integrated the DNA assembly method and the software with a robotic system named Illinois Biological Foundry for Advanced Biomanufatcuring (iBioFAB) to create a nearly fully automated workflow for PlasmidMaker. Our method allows assembly of fragments with GC-content as high as 77% and error-free assembly of plasmids as large as 27 kb including the ones containing multiple repeats from up to 10 DNA fragments. Only limited human interventions are required from design to build to test.

The overall end-to-end pipeline for PlasmidMaker is shown in Fig. 1. There are 4 major steps involved in the automated workflow for plasmid construction: (1) In the Design part, users can design plasmids with different DNA fragments in their preferred manner as well as search for common plasmids from a database to serve as their templates by using a frontend software. After an order is received, a technician can perform quality check by using a backend software. The sequences which pass the checking criteria can then be sorted into picklists and sent for construction. (2) In the Build part, according to the worklists generated, PCR plates for amplification of fragments are prepared. The Tecan FluentControl liquid handler is controlled by MomentumTM Workflow Scheduling software and F5 robotic arm to dilute and mix primers, templates, guides, and master mix into every single well of the 96-well plate. (3) In the Build part, one-pot automated PfAgo digestion, purification, ligation, and transformation of different plasmids are performed on the iBioFAB. (4) In the Test part, constructed plasmids by automated minipreps are verified through restriction digestion and gel electrophoresis. Correctly assembled plasmids are then re-cultured for making frozen stocks by the liquid handler. This high throughput pipeline allows completion of multiple plates of DNA assembly via standardized and integrated unit operations. Researchers only need to supervise the whole system while programmed software and robotic arms implement the complicated experiments.

In addition, we will provide plasmid construction as a service, where the customers can use the website to upload the plasmid sequences and request assembled plasmids from iBioFAB. Currently, we are rolling out the DNA assembly in different phases and we have started offering plasmid construction service to the researchers in the Department of Energy (DOE) Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) since March 2022. We will further offer this service to all DOE bioenergy research centers at the end of this year, followed by releasing it for ordering from the broad research community. Finally, since we are the founding member of the Global Biofoundry Alliance4, we will be more than happy to help other members to implement this PlasmidMaker pipeline in their institutions if they want.

Of note, even we have developed this tool as an alternative for constructing plasmids efficiently, the time and labor we spent to achieve this goal were tremendously huge. We still remember the multiple days and nights we went through for troubleshooting as well as optimizing the robotic workflows. We met weekly for more than 1.5 year just to establish the prototype. Fortunately, after all the difficult situations, we are currently able to provide a functional and user-friendly webpage that people can perform their designs specifically. More importantly, we will keep updating the website and optimizing our plasmid construction workflow according to different customers’ feedbacks and needs.

PlasmidMaker is a powerful platform consisting of advanced software and hardware for rapid construction of virtually any plasmids in a high throughput manner. It addresses a critical bottleneck in basic and applied biology and should greatly accelerate the development of synthetic biology for biotechnological and biomedical applications.

Fig. 1

Fig. 1. Our effort in developing PlasmidMaker for construction of plasmids from linear DNA parts via Design, Build, Test cycle and PfAgo/AREs.

Our paper:

Enghiad, B., Xue, P., Singh, N. et al. PlasmidMaker is a versatile, automated, and high throughput end-to-end platform for plasmid construction. Nat Commun 13, 2697 (2022).

Written by Pu Xue (Mason) and Huimin Zhao

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