Renders, M. | Search bioRxiv

bioRxiv2020

Towards XNA molecular biology: Bacterial cell display as a robust and versatile platform for the engineering of low affinity ligands and enzymes

Although directed evolution has been remarkably successful at expanding the chemical and functional boundaries of biology, it is limited by the robustness and flexibility of available selection platforms - traditionally designed around a single desired function with limited scope for alternative applications. We report SNAP as a quantitative reporter for bacterial cell display, which enabled fast troubleshooting and systematic development of the selection platform. In addition, we demonstrate that even weak interactions between displayed proteins and nucleic acids can be harnessed towards specific labelling of bacterial cells, allowing functional characterisation of DNA binding proteins and enzymes. Together, this establishes bacterial display as a viable route towards the systematic engineering of all ligands and enzymes required for the development of XNA molecular biology.

synthetic biology↗

bioRxiv2017

Efficiently Exploring Functional Space In Loop Engineering With Variations In Length And Composition

Insertions and deletions (indels) are known to affect function, biophysical properties and substrate specificity of enzymes, and they play a central role in evolution. Despite such clear significance, this class of mutation remains an underexploited tool in protein engineering with no available platforms capable of systematically generating or analysing libraries of varying sequence composition and length. We present a novel DNA assembly platform (InDel assembly), based on cycles of endonuclease restriction and ligation of standardised dsDNA building blocks, that can generate libraries exploring both composition and sequence length variation. In addition, we developed a framework to analyse the output of selection from InDel-generated libraries, combining next generation sequencing and alignment-free strategies for sequence analysis. We demonstrate the approach by engineering the well-characterized TEM-1 {beta}-lactamase {Omega}-loop, involved in substrate specificity, identifying multiple novel extended spectrum {beta}-lactamases with loops of modified length and composition areas of the sequence space not previously explored. Together, the InDel assembly and analysis platforms provide an efficient route to engineer protein loops or linkers where sequence length and composition are both essential functional parameters.

bioengineering↗