Bacterial Cell Display as a Robust and Versatile Platform for Engineering Low‐Affinity Ligands and Enzymes

Abstract

Directed evolution has been remarkably successful at expanding the chemical and functional boundaries of biology. That progress is heavily dependent on the robustness and flexibility of the available selection platforms, given the significant cost to (re)develop a given platform to target a new desired function. Bacterial cell display has a significant track record as a viable strategy for the engineering of mesophilic enzymes, as enzyme activity can be probed directly and free from interference from the cellular milieu, but its adoption has lagged behind other display-based methods. Herein, we report the development of SNAP as a quantitative reporter for bacterial cell display, which enables fast troubleshooting and the systematic development of the display-based selection platform, thus improving its robustness. In addition, we demonstrate that even weak interactions between displayed proteins and nucleic acids can be harnessed for the specific labelling of bacterial cells, allowing functional characterisation of DNA binding proteins and enzymes, thus making it a highly flexible platform for these biochemical functions. Together, this establishes bacterial display as a robust and flexible platform, ideally suited for the systematic engineering of ligands and enzymes needed for XNA molecular biology.

Absolute protein quantification using fluorescence measurements with FPCountR

“…Further, such absolute quantification need not be limited to fluorescent proteins. The last few years have seen a fantastic expansion of fluorogenic molecules, tools that have allowed the specific quantification of localised proteins [47][48][49] , proteins in anaerobic environments 50,51 , and the fluorescent quantification of RNAs 44,52,53 . Calibration of these molecules would be more complex to achieve but no less valuable.…”

FPCountR: Absolute protein quantification using fluorescence measurements

“…Absolute quantification need not be limited to fluorescent proteins. The last few years have seen a fantastic expansion of fluorogenic molecules, tools that have allowed the specific quantification of localised proteins (Svendsen et al ., 2008; Li et al ., 2018; Csibra et al ., 2020), proteins in anaerobic environments (Streett et al ., 2019; Charubin et al ., 2020), and the fluorescent quantification of RNAs (Pothoulakis et al ., 2014; Siegal-Gaskins et al ., 2014; Yerramilli and Kim, 2018). Calibration of these molecules would be more complex to achieve but no less valuable.…”

Controlled E. coli Aggregation Mediated by DNA and XNA Hybridization

“…Interestingly, SNAP-tag technology has barely been used in bacterial systems and was mainly dedicated to the capture of environmental molecules, [43] bioimaging, [44] and bacterial cell display. [45] Covalent surface immobilization of oligonucleotides via SNAP-tag conjugation allows inducing more robust artificial contacts between nonadherent cells. The attachment of the chemically modified oligonucleotides at the cell surface is inducible, selective, and covalent but the aggregation itself is non-covalent and more-over reversible.…”

“…To do so, we genetically engineered E. coli to express the protein SNAP‐ tag at the cell surface and subsequently conjugated it to both complementary unmodified and modified oligonucleotides at the surface of two distinct cell populations forming a bacterial tissue. Interestingly, SNAP‐ tag technology has barely been used in bacterial systems and was mainly dedicated to the capture of environmental molecules, [43] bioimaging, [44] and bacterial cell display [45] . Covalent surface immobilization of oligonucleotides via SNAP‐tag conjugation allows inducing more robust artificial contacts between non‐adherent cells.…”