A modular HaloTag platform for engineering magnetically responsive bacterial microrobots

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.