Current protein screening technologies pose a significant bottleneck in our ability to evolve and engineer new protein functions due to their low throughput and inability to place proteins into their native structure or expose them to solutions containing targets or analytes. To address these limitations, we have developed PicoShell technology, a cell and protein screening tool that allows scientists to screen millions of variants while sequentially exposing proteins to test their function in varying solutions relevant to their end application. PicoShells can be coupled with fluorescence-activated cell sorting (FACS) to screen and sort millions of variants with throughputs of >50 variants per second.
To demonstrate the potential of PicoShell technology as a viable platform to evolve proteins, we aim to evolve fluorescent protein glucose biosensors. We have collected preliminary data with protein calcium biosensors, a sensor that is more thoroughly developed and is a better system for the initial optimization of the workflow. Clonal colonies of E. coli expressing GCaMP, a protein with fluorescence intensity that is modulated by calcium concentration, can be lysed inside PicoShells to release proteins. The outer shell is engineered to have a MWCO <15 kDa, enabling PicoShells to retain GCaMP sensors from lysed cells. Retained GCaMP sensors fluoresce proportionally to changes in calcium concentrations. Variants with higher or lower fluorescent readouts can be sorted and genetic material encoding the selected variant can be amplified via PCR and prepared for sequencing.
To expand the capabilities of the PicoShell workflow, we are working with BioPACIFIC’s BioFoundry Core at UCLA to integrate the PicoShell workflow with their automation workflows, which can significantly increase the number of variants that can be amplified and sequenced post-sort. By collaborating with the BioFoundry Core, we aim to improve our workflow’s scalability and accessibility, enabling others to evolve new proteins with dramatically reduced timelines and costs.