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

Enghiad, Behnam; Xue, Pu (ORCID:0000000160231298); Singh, Nilmani (ORCID:0000000236847022); Boob, Aashutosh Girish; Shi, Chengyou (ORCID:0000000311383580); Petrov, Vassily Andrew; Liu, Roy; Peri, Siddhartha Suryanarayana; Lane, Stephan Thomas; Gaither, Emily Danielle; Zhao, Huimin (ORCID:0000000290696739)

doi:10.1038/s41467-022-30355-y

DOE OSTI · 1867945

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

Enghiad, Behnam·Xue, Pu (ORCID:0000000160231298)·Singh, Nilmani (ORCID:0000000236847022)·Boob, Aashutosh Girish·Shi, Chengyou (ORCID:0000000311383580)·Petrov, Vassily Andrew·Liu, Roy·Peri, Siddhartha Suryanarayana·Lane, Stephan Thomas·Gaither, Emily Danielle·Zhao, Huimin (ORCID:0000000290696739)

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Abstract

Abstract Plasmids are used extensively in basic and applied biology. However, 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. Here, we report the development of a versatile, robust, automated end-to-end platform named PlasmidMaker that allows error-free construction of plasmids with virtually any sequences in a high throughput manner. This platform consists of a most versatile DNA assembly method using Pyrococcus furiosus Argonaute ( Pf Ago)-based artificial restriction enzymes, a user-friendly frontend for plasmid design, and a backend that streamlines the workflow and integration with a robotic system. As a proof of concept, we used this platform to generate 101 plasmids from six different species ranging from 5 to 18 kb in size from up to 11 DNA fragments. PlasmidMaker should greatly expand the potential of synthetic biology.

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59 BASIC BIOLOGICAL SCIENCES expression systems genetic techniques synthetic biology

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Enghiad, Behnam, Xue, Pu (ORCID:0000000160231298), Singh, Nilmani (ORCID:0000000236847022), Boob, Aashutosh Girish, Shi, Chengyou (ORCID:0000000311383580), Petrov, Vassily Andrew, Liu, Roy, Peri, Siddhartha Suryanarayana, Lane, Stephan Thomas, Gaither, Emily Danielle, Zhao, Huimin (ORCID:0000000290696739). 2022-05-16. PlasmidMaker is a versatile, automated, and high throughput end-to-end platform for plasmid construction. https://doi.org/10.1038/s41467-022-30355-y

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