Bioprinting horizontal gene transfer: a bacterial “photocamera” SECTION 1: ABSTRACT Horizontal gene transfer (HGT) via bacterial conjugation is a primary mechanism driving the dissemination of antibiotic resistance genes in clinical and environmental microbial communities, yet the spatial dynamics of conjugative transfer remain poorly visualized in real time. This project proposes to engineer a mobilizable donor plasmid encoding the bglA β-glucosidase reporter gene from Enterococcus faecalis, synthesized as a Clonal Gene from Twist Bioscience (pTwist Amp High Copy backbone), and to deploy it in a triparental mating system using E. coli DH5α as both donor and helper strains and E. coli K-12 wild-type as the recipient. Gene transfer is spatially visualized on UTI chromogenic agar, which contains dual chromogenic substrates for β-galactosidase and β-glucosidase: the recipient K-12 strain (LacZ⁺) produces pink colonies, the donor DH5α (BglA⁺, LacZ⁻) produces teal colonies, and transconjugants expressing both enzymes produce a distinctive purple/lilac color that is absent from either parental strain alone. The Opentrons OT-2 robotic liquid handler deposits all three strains in geometrically precise concentric overlapping circles onto plain LB agar for conjugation, and subsequently onto UTI chromogenic agar for spatial readout. Transconjugant identity is confirmed by colony PCR targeting both the plasmid-encoded bglA and the chromosomal lacZ of K-12. This project establishes a proof-of-concept platform for the spatial mapping of plasmid transfer events, providing a visual, automation-compatible, and instrument-free assay for studying conjugation dynamics with direct relevance to antibiotic resistance surveillance and the design of synthetic microbial communities.