Recombinant expression systems are essential tools, widely employed in both industrial applications and academic research. However, as researchers tackle increasingly complex and challenging problems, existing expression systems are often inadequate. Recombinant protein production, biosensors, and synthetic pathways depend on efficient delivery of multiple genes into a cell. This challenge becomes substantially more difficult when working with more than three genes, presenting a major barrier to the production of e.g. multi-component complexes. In this work, we combine several innovations from diverse technologies to address this hurdle. Our tailored mammalian vector system, "bigMAC", is designed to support the co-expression of numerous genes. BigMAC is a transient expression system, featuring modular components that facilitate rapid replacement and reassembly. By re-purposing optimised flank regions, bigMAC facilitates the straightforward Gibson assembly of up to five genes into a single vector. By integrating two orthogonal counter-selection technologies, BigMAC removes inefficiencies from cloning workflows. Furthermore, this enables the hierarchical assembly of multiple five-component vectors into higher-order constructs. Thus, our technology simplifies complex assembly workflows, enhances scalability in mammalian systems, and unlocks challenging experiments.
Correspondence: charles.bayly-jones@monash.edu, andrew.ellisdon@monash.edu