Sortase-Mediated Labeling of M13 Bacteriophage and the Formation of Multi-Phage Structures

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Sortase-Mediated Labeling of M13 Bacteriophage and the Formation of Multi-Phage Structures

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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.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

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