Background

Molecular cloning is a hallmark of modern biotechnology with the ability to repurpose recombinant DNA into a variety of genetic circuits that can represent a spectrum of purposes. However, a major limitation in exploring the permutations possible in construction of genetic circuits is the ability to rapidly prototype, test and implement improvements on circuit designs. For this to come to fruition, the time from designing genetic circuits to delivery into cells needs to be expedited from conventional cloning methods, such as Gibson cloning (Akama-Garren et al., 2016) or restriction digests. We designed a framework in which a conventional gene circuit is deconstructed into its constituent components such that one can easily swap these components to quickly assemble vast combinations, assessing how each iteration affects function. Inspired by earlier iterations of cloning toolkits (Weber et al., 2011a and 2011b; Duportet et al., 2014; Lee et al., 2015; Martella et al., 2017; Pérez-González et al., 2017; Halleran et al., 2018; Pollak et al., 2019), we adopted a Golden-gate cloning system where assembly of constituent parts into functional units can be accomplished in a one-pot fashion (Weber et al., 2011a and 2011b). To concretely realize the promise of this framework, we built and characterized over 400 DNA parts that encode a myriad of reagents. We named the curated library and cloning framework the Mammalian ToolKit (MTK) (Fonseca et al., 2019). With the MTK, swapping promoters, switching fluorophores, or rapidly prototyping protein tags constitutes changing one plasmid in a reaction. This is markedly different from having to redesign oligos and PCR verifying correct assembly for every use case, thus discouraging continued optimization of a construct of interest. The hierarchical nature of this system enables a library of parts to expand to a library of transcriptional units (TUs) that can be further combined to create multi-transcriptional unit vectors that are then delivered to cells. These TUs are delivery-agnostic, promoting their multiple uses. For example, the same TUs used in a circuit delivered to a specific locus via CRISPR-Cas9 homologous recombination can be recycled in a PiggyBac transposase transfection to be randomly integrated. While the initial time from part verification to delivery into cells is comparable to conventional cloning (approximately 4 days), the repurposing of parts and TUs allows vast combinations of circuits to be assembled in only 2 days.

Despite the tremendous advantages the MTK presents, the barrier to entry may still dissuade those without a cloning background. To ameliorate the activation energy for adoption across all disciplines, here we present instructions on how to utilize the current library to assemble novel circuits, and a step-by-step protocol to “domesticate” new parts (that is to make them compatible with Golden Gate-based assembly of the MTK), assemble Transcriptional Units (TU), and create varied multi-transcriptional units (multi-TU). It is our goal to make this toolkit widely accessible and available to enhance the development of mammalian expression systems towards new and exciting discoveries in biology.