Gearbox Biosciences - Antibiotic-Free Recombinant Protein with Pop-Out-Plasmid Technology
Gearbox Biosciences, a spin-off from the University of Tartu, Estonia, is pioneering antibiotic-free recombinant protein production through its innovative Pop-Out-Plasmid Technology. This advanced system eliminates the need for traditional selection markers like antibiotics and chemical inducers such as IPTG in E. coli cultures. By integrating the gene of interest directly into the host chromosome, our technology allows for stable maintenance during biomass growth. The subsequent production phase is initiated by a simple temperature increase, inducing a batch of molecular events that result in robust protein expression with increased scalability and efficiency. This decoupling of growth and production stages enhances yield while minimizing environmental risks associated with antibiotic use. Our work is supported by strategic collaborations and grants, allowing us to extend our technology for plasmid DNA production, critical in the realm of gene and cell therapy.
In Pop-Out-Plasmid™ Technology the gene of interest is integrated as a single copy into the host chromosome and therefore is stably maintained during the biomass growth stage at 30°C. At the desired time the production stage can be switched on by temporarily increasing the culture temperature to 37°C. This triggers a series of events at the molecular leveL
Cell division is stopped by excision of the origin of replication. oriC from the bacterial chromosome. The mechanism is the basis of our patent pending Switcher Technology, in which the Switched cells do not divide but remain metabolically active. This decouples protein production from cellular growth and increases protein expression. Please find more details about Switcher Technology from our research published in ACS Synthetic Biology here.
The gene of interest together with other genetic elements is excised from the chromosome and forms a replicating plasmid. The copy number of this plasmid increases because a plasmid-specific origin of replication becomes active. Consequently, this results in a high protein expression LeveL Most importantly, the production process is antibiotic-free, because plasmids cannot be lost from non-dividing cells.
Protein expression can be induced by relocating a promoter in front of the encoding gene during plasmid formation. No chemical inducer is needed.
The animated video below will give a better visual overview of the Pop-Out-Plasmid Technology and its key benefits.