A modular HaloTag platform for engineering magnetically responsive bacterial microrobots

Abstract Functionalizing bacteria with magnetic nanoparticles (MNPs) is a common route toward magnetically responsive bacterial microrobots. However, existing strategies are often limited by low functionalization efficiency, weak binding, poor reproducibility, and nonspecific interactions. Here, we present a robust, specific, and reproducible magnetic functionalization platform based on the self-labeling protein HaloTag, yielding magnetic bacterial microrobots termed HaloBots. Using Escherichia coli as an engineering host, HaloTag was displayed on the outer membrane via the Lpp–OmpA anchoring system, with the assistance of a Long and Flexible (LF) peptide linker. Optimization of genetic engineering vectors and colony purification enabled robust and uniform HaloTag display across bacterial populations. In addition, MNPs were modified with chloroalkane–PEG ligands to enable site-specific covalent conjugation with HaloTag. Tuning the ligand density on MNPs revealed a critical balance between ligand accessibility and surface charge for achieving efficient and specific attachment. Consequently, the resulting HaloBots exhibited stable MNP conjugation and reliable magnetic actuation. Collectively, this work establishes a modular and tunable strategy for engineering magnetically responsive bacterial microrobots.