DOI: 10.1101/506188Dec 26, 2018Paper

A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells

BioRxiv : the Preprint Server for Biology
Joao Pedro FonsecaHana El-Samad

Abstract

The ability to rapidly assemble and prototype cellular circuits is vital for biological research and its applications in biotechnology and medicine. Current methods that permit the assembly of DNA circuits in mammalian cells are laborious, slow, expensive and mostly not permissive of rapid prototyping of constructs. Here we present the Mammalian ToolKit (MTK), a Golden Gate-based cloning toolkit for fast, reproducible and versatile assembly of large DNA vectors and their implementation in mammalian models. The MTK consists of a curated library of characterized, modular parts that can be easily mixed and matched to combinatorially assemble one transcriptional unit with different characteristics, or a hierarchy of transcriptional units weaved into complex circuits. MTK renders many cell engineering operations facile, as showcased by our ability to use the toolkit to generate single-integration landing pads, to create and deliver libraries of protein variants and sgRNAs, and to iterate through Cas9-based prototype circuits. As a biological proof of concept, we used the MTK to successfully design and rapidly construct in mammalian cells a challenging multicistronic circuit encoding the Ebola virus (EBOV) replication complex. This c...Continue Reading

Related Concepts

Biotechnology
Ebola virus
Genetic Vectors
Research
Transcription, Genetic
Virus Replication
Virus
RNA, Guide
Buccal Fat Pad
Inhibitors

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