dc.contributor.advisor | Belcher, Angela | |
dc.contributor.author | Hess, Gaelen | |
dc.date.accessioned | 2012-11-15T15:52:30Z | |
dc.date.issued | 2012-11-15 | |
dc.date.submitted | 2012 | |
dc.identifier.citation | Hess, Gaelen. 2012. Sortase-Mediated Labeling of M13 Bacteriophage and the Formation of Multi-Phage Structures. Doctoral dissertation, Harvard University. | en_US |
dc.identifier.other | http://dissertations.umi.com/gsas.harvard:10513 | en |
dc.identifier.uri | http://nrs.harvard.edu/urn-3:HUL.InstRepos:9909631 | |
dc.description.abstract | M13 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_US |
dc.language.iso | en_US | en_US |
dash.license | LAA | |
dc.subject | M13 bacteriophage | en_US |
dc.subject | nanostructures | en_US |
dc.subject | sortase | en_US |
dc.subject | biophysics | en_US |
dc.title | Sortase-Mediated Labeling of M13 Bacteriophage and the Formation of Multi-Phage Structures | en_US |
dc.type | Thesis or Dissertation | en_US |
dc.date.available | 2012-11-15T15:52:30Z | |
thesis.degree.date | 2012 | en_US |
thesis.degree.discipline | Biophysics | en_US |
thesis.degree.grantor | Harvard University | en_US |
thesis.degree.level | doctoral | en_US |
thesis.degree.name | Ph.D. | en_US |
dc.contributor.committeeMember | Hu, Evelyn | en_US |
dc.contributor.committeeMember | Shih, William | en_US |
dc.contributor.committeeMember | Ploegh, Hidde | en_US |
dc.contributor.committeeMember | Hogle, James | en_US |
dc.contributor.committeeMember | Springer, Timothy | en_US |