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

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 construct provides a non-infectious biosafety level 2 (BSL2) cellular assay for exploring the transcription and replication steps of the EBOV viral life cycle in its host. Its construction also demonstrates how the MTK can enable important and time sensitive applications such as the rapid testing of pharmacological inhibitors of emerging BSL4 viruses that pose a major threat to human health..

Choice of selectable marker affects recombinant protein expression in cells and exosomes

“…Many researchers have devoted significant time, effort, and resources to improving the outcomes of mammalian cell transgenesis experiments. Much of this effort has been directed at improved vector design, resulting in the identification of many cis -acting features that affect transgene expression, including transcriptional regulatory regions ( 6 , 7 , 8 , 9 , 10 ), mRNA polyadenylation (pAn) sites ( 11 ), introns ( 12 ), mRNA export and/or translation signals ( e.g. , the Woodchuck hepatitis virus [WHP] posttranscriptional regulatory element [WPRE] ( 13 )), and cis -acting inhibitors of gene silencing ( 10 , 14 ).…”

“…Much of this effort has been directed at improved vector design, resulting in the identification of many cis -acting features that affect transgene expression, including transcriptional regulatory regions ( 6 , 7 , 8 , 9 , 10 ), mRNA polyadenylation (pAn) sites ( 11 ), introns ( 12 ), mRNA export and/or translation signals ( e.g. , the Woodchuck hepatitis virus [WHP] posttranscriptional regulatory element [WPRE] ( 13 )), and cis -acting inhibitors of gene silencing ( 10 , 14 ). Other studies have revealed that transgene expression can be improved by eliminating or reducing bacterial or viral sequences that may induce transgene silencing ( 15 , 16 ), such as by use of DNA minicircles ( 17 ) or DNA transposons ( e.g.…”

Versatile Labeling and Detection of Endogenous Proteins Using Tag-Assisted Split Enzyme Complementation

“…We generated 'part' vectors, 'expression' vectors, and 'landing pad' vectors following the Mammalian Toolkit (MTK) approach 28 .…”

“…The split-GFP, and split-Halo Landing Pad HEK293Ts, were generated from a published landing pad parent cell line 28 seeded at 100k cells in a 12-well plate. To each well, 600 ng of BxbI Integrase Expression Vector (Addgene #51271) and 600 ng of each landing pad donor plasmid were co-transfected.…”

Improved betulinic acid biosynthesis using synthetic yeast chromosome recombination and semi-automated rapid LC-MS screening

“…Synthetic biology now offers many tools to rapidly modify pathways and genomes to optimise metabolite titres. For example, modular DNA pathway construction methods are ubiquitous and now available for a variety of microorganisms [5][6][7][8][9][10] . The nascent field of synthetic genomics also now allows entirely new chromosome designs that introduce new tools for genome optimisation for metabolic engineering purposes.…”