Belcher, AngelaHess, Gaelen2012-11-152012-11-152012Hess, Gaelen. 2012. Sortase-Mediated Labeling of M13 Bacteriophage and the Formation of Multi-Phage Structures. Doctoral dissertation, Harvard University.http://dissertations.umi.com/gsas.harvard:10513http://nrs.harvard.edu/urn-3:HUL.InstRepos:9909631M13 filamentous bacteriophage has been used as a biotemplate for the nucle- ation of materials. Phage is an ideal and diverse scaffold with its large aspect ratio and ability to display biomolecules to bind a range of targets. To form more complex patterned materials, interactions between the phage must be specific and reliable. We develop a phage labeling method using sortase enzymes to create multi-phage nanostructures. We exploit two sortases and functionalize the N-termini of the pIII, pIX, and pVIII proteins with small and large moieties. For the pVIII, we show a 100 fold improvement in display of GFP molecules on the phage surface. Taking advantage of orthogonal sortases, we simultaneously label two capsid proteins on a single phage particle. Using these N-terminal labeling techniques, we demonstrate fluorescent staining of cells and construct a lampbrush phage structure linking the pIII of one phage to the pVIII of another using a biotin-streptavidin linkage. To further expand our labeling repertoire, C-terminal sortase labeling of phage was pursued. To achieve this goal, we transfer a loop structure from cholera toxin to pIII and label it with a fluorophore and a multi-domain protein. With this archi- tecture, we form end-to-end dimers using sortase to conjugate the loop structure to phage containing the nucleophile motif. Lastly, we investigate DNA hybridization as a method for crosslinking phage. Using sortase, we label the pVIII on two sets of phage: one with ssDNA and the other with a complementary DNA oligonucleotide. We anneal these phages together and observe phage networks that are dispersed by heat and reform upon cooling.en-USM13 bacteriophagenanostructuressortasebiophysicsThesis or Dissertation2012-11-15