Genetic Engineering of Functional Large Amyloid Fibers

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dc.contributor.authorRoth, David Eugeneen
dc.contributor.committeechairBarone, Justin R.en
dc.contributor.committeememberRuder, Warren Christopheren
dc.contributor.committeememberDavalos, Rafael V.en
dc.contributor.departmentBiological Systems Engineeringen
dc.date.accessioned2017-07-23T06:00:13Zen
dc.date.available2017-07-23T06:00:13Zen
dc.date.issued2016-01-29en
dc.description.abstract"Template" and "adder" proteins can be genetically encoded to produce large amyloid fibers when mixed together. Escherichia coli is used to clone a "template" protein, Gd20, which will cooperatively self-assemble with two "adder" proteins, P7Q and P7S, to yield two different large amyloid fibers. Atomic force microscopy (AFM) is used to image the fibers and AFM tip approach/retraction force is used to quantify molecular packing in the fibers. Glutamine (Q)-containing P7Q and serine (S)-containing P7S both have the same hydrophobic core, charge, and hydrogen bonding potential. However, P7Q is highly alpha-helical while P7S contains a beta-sheet core. After 72 hours, the Gd20:P7Q template:adder protein mixture produces tightly packed ~0.3 μm high and ~1.9 μm wide fibers that exhibit a low retraction force of ~44 nN after indentation. The Gd20:P7S mixture produces larger ~1.1 μm high and ~9.7 μm wide fibers exhibiting a much higher retraction force of ~503 nN showing they are much less molecularly packed. These results indicate that the adder protein alpha-helical character is important for self-assembly and molecular packing inside of the large amyloid fiber. The experimental results show that large amyloid fibers with predictable size and mechanical properties can be anticipated and encoded at the genetic level.en
dc.description.degreeMaster of Scienceen
dc.format.mediumETDen
dc.identifier.othervt_gsexam:7136en
dc.identifier.urihttp://hdl.handle.net/10919/78399en
dc.publisherVirginia Techen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectself-assemblyen
dc.subjectgenetic encodingen
dc.subjectfibrilen
dc.subjectfiberen
dc.subjectamyloiden
dc.subjectcellular expressionen
dc.subjectpoint spectroscopyen
dc.subjectnanoindentationen
dc.subjectmolecular packingen
dc.subjectretraction forceen
dc.subjectAFMen
dc.titleGenetic Engineering of Functional Large Amyloid Fibersen
dc.typeThesisen
thesis.degree.disciplineBiological Systems Engineeringen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

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