purcc 2012 - University of the Pacific

Poster Session Abstracts
Exploration of Pyriform Spidroin1
(PySp1) QEA Module’s Role in Fiber
Formation and Properties
Cynthia Co Ting Keh, Frances Pham,
Yifeng Li, Steve Oh
Faculty Mentor: Craig Vierra
Spider silk, known for its tensile strength,
extensibility and toughness, has a wide array of
potential uses that span from medical equipment
to military armor. Spider silk’s strength is
comparable to high-tensile steel, yet it can
withstand considerable strain or extensibility
without breaking. There are several types of
spider silk, each originating from a different silkproducing
gland. These fibers provide different
functions for spiders, ranging from web
constructions to locomotion. Pyriform spidroin 1
(PySp1), the protein under study, was first
identified in the pyriform gland, whose main
function is to produce silk that is used to
immobilize dragline silk. Our research focused
on the significance of PySp1 QEA module and
its relationship to the mechanical adhesive
properties of pyriform silks. The PySp1 cDNA
encoding the QEA module was amplified from a
cDNA library prepared from the silk-producing
glands of the black widow spider, Lactrodectus
hesperus. After insertion of the cDNA into a
prokaryotic expression vector, we transformed E.
coli and performed a restriction digestion
analysis to check for the cDNA insert presence
and correct directionality. Following the
validation of the presence of a cDNA insert, the
QEA PySp1 cDNA was induced and its product
monitored through both blue silver staining and
western blotting. Western blot analysis showed
that QEA PySp1 was expressed in high levels.
To further understand its role in pyriform silk
formation and function, we plan to purify the
protein through nickel affinity chromatography.
We hope to spin fibers from the purified protein
solution in order to better comprehend its
mechanical properties and function.
Studying the Function of Pyriform
Spidroin-2 Modules
Eugene Kim, Mariah Mayo, Katherine Yin
Faculty Mentor: Craig Vierra
Extensive research on the properties of spider
silk has sparked the interests of the general
community in discovering potential uses for
these materials. Mechanical properties have
shown that the strength of spider silk is
comparable to that of high-tensile steel. When
comparing other properties, spider silk is more
extensible and tougher relative to high-tensile
steel and Kevlar. With such superb mechanical
properties, spider silk is being further studied for
future industrial uses.
Pyriform Spidroin 2 (PySp2) is a glue silk
fibroin spun by orb-weaving spiders from the
pyriform gland. PySp2 is spun into attachment
discs as a viscous liquid that dries rapidly,
allowing for the fastening of dragline silk, which
facilitates locomotion and web construction. The
internal block-repeat sequences of PySp2 have
the ability to self-assemble, promoting fiber
formation to occur in a liquid environment. The
PySp2 protein sequences play a central role in
affecting the protein’s overall mechanical
properties.
The goal of our research was to express
truncated versions of PySp2 proteins in bacteria
and spin fibers from these purified proteins in
order to test its mechanical properties. To
express the proteins, the PySp2 cDNA was
inserted into the cloning vector pBAD-Thio-
TOPO, which was propagated in bacteria.
Bacterial cells carrying the expression vector
were induced and the resulting recombinant
proteins were further studied. Western blot
analysis was used to verify the expression of the
protein.
Results showed that PySp2 recombinant proteins
were expressed in bacteria, specifically in cells
carrying the expression vectors with the PySp2
cDNA. These positive results will allow further
experimentation and observation of the
mechanical properties of glue silk fibers spun
from the purified proteins.
Expression of an Internal Block Repeat
Domain from Pyriform Spidroin1, a Glue
Silk Protein from Black Widow Spiders
Jay Ko, Nancy Nguyen, Raymond Pandez,
Vu Tran
Faculty Mentor: Craig Vierra
Spider silk is composed of proteins that allow for
its high extensibility and tensile strength, making
spider silk an ideal substance for stitching
wounds, bandaging, and other medical uses. By
synthetically producing spider silk proteins in
vitro, ample amounts of spider silk can be
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