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BIOL 536: Spider Silk Presentation

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Hilary Andrews

on 6 May 2013

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Transcript of BIOL 536: Spider Silk Presentation

Spider Silk Background Introduction Materials
& Methods Results Discussion Questions? Group 1 Presentation Hilary andrews
Rey Ayon
Nghi Do
Ryon Seymore
Madison Smith After the proteins were separated they were moved to polyvinylidene difluoride membranes

Antibodies were added for immunodetection

The antigen-antibody complexes that resulted were visualized using chemiluminescence

The reactions were then quantified with known amounts of the chimeric silkworm/A2S814 spider silk protein from E. coli as standards Silk glands homogenized on ice in sodium phosphate buffer containing 1% SDS and 5 M urea

Mixture clarified in microcentrifuge for 5 minutes at 4 degrees C

The supernatants(liquid portion) were harvested as silk gland extracts

Silk gland extracts and sequential cocoon extracts from earlier were diluted 4x with 10 mM of Tris·HCl/2% SDS/5% BME buffer, and the samples that contained

~ 90 μmicrograms of total protein were mixed 1:1 with SDS/PAGE loading buffer and boiled at 95 degrees C and then loaded on to 4 – 20 % gradient gels Analysis of Silk Proteins Soluble and insoluble separated again and soluble stored at -20 degrees C

Insoluble pellet resuspended in 1 mL of 9 M LiSCN containing 2% βBeta-mercaptoethanol (BME) and incubated for 16-48 hours at room temperature

After separation, soluble stored at -20 degrees C

Insoluble pellet resuspended in 1 mL of 16 M LiSCN containing 5% BME and incubated for 1 hour at room temperature

This resulted in complete dissolution and produced the final extract which was held at 20 degree C

Immunoblotting assays done on all extracts Degummed proteins under went protein extraction

30 mg of degummed silk fiber from cocoons produced by two individuals in each line were treated with 1 mL of phosphate buffered saline for 16 hours at 4 degrees C

Material separated into soluble and insoluble portions via centrifugation

Soluble portion removed and stored at -20 degrees C

Insoluble pellet resuspended in 2% sodium dodecyl sulfate (SDS) for 16 hours at room temperature Parental and transgenic silkworm cocoons were harvested and sericin (gummy protein) layer removed

Degummed silk fibers were washed twice with hot water

Fibers then freeze dried and weighed to determine how good the sericin removal was Sequential Extraction of Silkworm Cocoon Proteins After hatching, larvae were reared on an artificial diet

Subsequent generations obtained by mating siblings in the same line

Transgenic progeny identified by the presence of a fluorescent eye marker Eggs collected one hour after they were laid and arranged on a slide

Preblastoderm embryos injected with 1–5 nL of vector and helper plasmid DNA mixtures dissolved in injection buffer

Punctured eggs sealed with Helping Hand Super Glue gel and incubated Silkworm Transformation Glands then placed in Bio-Rad Biolistic PDS-1000/He Particle Delivery System chamber

Glands were bombarded

After bombardment, glands placed in Grace’s medium with 2x antibiotics and incubated at 28 degrees C

Transient expression assessed by looking for markers with fluorescence microscopy

Immunoblotting testing done too B. mori silkworms sterilized with 70% alcohol on the third day of their fifth instar

Entire silk glands aseptically dissected and placed in Grace’s medium plus antibiotics

3 mg aliquots of tungsten microparticles covered in 5 µg of the relevant piggybac DNA

Glands transferred to a new medium containing 1% sterile agar Ex Vivo Silk-Gland
Bombardment Assay piggyBac Vector Constructions The degummed silkworm and spider silk fibers used for testing were initially 19 mm long

The average cross-sectional diameters were measured across two brins comprising the degummed silkworm silk and dragline spider silk fibers.

Single-fiber testing done with MTS Synergie 100 system

Mechanical data was recorded at a strain rate of 5 mm/min and the frequency of 250 MHz, which allowed for the calculation of stress and strain values Mechanical Testing of
Silk Fibers Regulatory elements and protein-coding sequences assembled into functional cassettes of two intermediate plasmids
(B and C)

Cassettes excised and subcloned to produce two piggybac vectors (D) Genomic DNA isolated from silk glands and produced with PCR

Each amplification product gel-purified and recovered

Cloned into plasmid vectors and bacteria that were successfully transformed were identified Bombix mori Analysis in this study was possible because one of the piggyBac vectors constructed encoded the same chimeric silkworm/spider silk protein, but with an enhanced
green fluorescent protein
(EGFP) tag.




Ex vivo silk-gland
bombardment assays to examine chimeric spider silk protein expression
in silk glands. What was the association of the
chimeric silkworm/spider silk proteins
with the composite silk fibers
produced by the
transgenic silkworms? Analysis of the Composite
Silk Fibers Result: Assay results showed that the
GFP-tagged silkworm/spider silk protein
induced green fluorescence
in the posterior silk gland region.










This meant that the basic design of
the piggyBac vectors worked and that
the experiment could continue. Transgenic silkworms encoding synthetic spider silk proteins
can, indeed, spin composite silk fibers with improved mechanical properties, relative to the fibers produced by the parental animals. Results of Toughness
Portrayed Graphically The composite fibers containing chimeric silkworm/spider silk proteins were significantly tougher than parental silkworm fibers and as tough as native dragline spider silk fibers.

Transgenic silkworms were more extensible than both the parental silkworm and native dragline spider silks Mechanical Properties of the Composite Silk Fibers No proteins detected in pnd-w1 silk fibers (lanes 3–-6)

One immunoreactive protein detected for spider 6 fibers (lane 11)
~106 kDa

Two immunoreactive proteins detected for spider 6-GFP fibers (lane 16)
~130 and ~110 kDa Visual inspection & immunoblotting of cocoon extracts

Result: Showed that they had isolated transgenic silkworms encoding the chimeric silk protein and that these proteins were associated with the silk fibers produced by those transgenic animals. Transgenic Silkworm
Isolation Result: Gel electrophoresis showed the fiber samples from the transgenic silkworm silk weighed more, which meant they indeed were made of a combination of silkworm/spider silk protein. Use silkworms to spin the spider silk proteins.

Because silkworms naturally spin silk, the silk will be a combination of spider silk and silkworm silk.

It is expected that the composite silk fibers spun by the transgenic silkworms will have improved mechanical properties, relative to the fibers spun by the parental silkworms. Solution Such hosts used to produce the spider silk proteins did not provide high yields.

In addition, the spinning of the silk proteins into fibers is a complex process that could not be done by the hosts used. Problems Silk genes of Nephila clavipes and the proteins they encode are repetitive.

There is a correlation between the repetitive amino acid sequences of the silk proteins and the mechanical properties of the silk fibers.

The genes for these spider silk proteins were used to encode in various hosts such as bacteria, yeast, bacilovirus/insect systems, mammalian cells, transgenic plants and transgenic animals. Spider Silk Proteins Silkworm silk is used now, particularly for surgical sutures, but spider silk has superior physical properties that make it more ideal for the finer sutures such as ocular, neurological, and cosmetic surgeries.

Farming silkworms to mass produce silkworm silk is easier and less costly than farming spiders for their silk.

Spider farming is prevented by the territorialism and cannibalism among the species. Silkworm Silk versus Spider Silk Potential biomaterials for:
wound dressings
artificial ligaments and tendons
tissue engineering
microcapsules
And more! Why silk fibers? Attributions to mechanical
properties:


Increased silk fiber toughness.

Usage of a 2.4kbp A2S814 synthetic spider silk sequence encoding repetitive flagelliform–like elastic and major ampullate motifs. Significance of this study:

Transgenic silkworms producing composite silk fibers with stably integrated chimeric silkworm/spider silk proteins that significantly improved the mechanical properties when compared to parental silks. Previous studies:

Usage of fhc promoters, but incorporating short peptide sequences of “silk-like” proteins.




Small size of silk peptides = minor changes to mechanical properties of silk fibers. Fibroin heavy chain (fhc) promoter = targets expression of foreign spider silk protein to the posterior silk gland.

fhc enhancer = increases silk protein expression levels. Silkworm properties:

piggyBAC vector assembly allows targeting of foreign gene expression in a silk gland-specific fashion. Discussion Bombyx mori Silkworms as ideal platforms for recombinant spider silk fiber manufacturing. Spider cannibalism
/ territoriality Nephila clavipes Constraints in spider farming for selectively manufacturing heterogeneous spider silks. Attributions to mechanical properties :

Usage of fhc promoter allows for expression of recombinant silk genes in concert with endogeneous silkworm fhc activity, the fibroin light chain, and fibrohexamerin proteins in the posterior silk gland. Attributions to mechanical properties :

A2S814 spider silk sequence has a naturally high propensity to form stably consolidated silk fibers. Previous studies:

Failure to use fhc promoter.




Usage of Ser1 promoter targets expression of recombinant silk proteins to middle silk gland.




Minor changes to mechanical properties of silk fibers. Silkworm properties: Silkworm properties:

Easy transformation of silkworms via piggyBAC vector usage. Spider silks are instrumental as functional biomaterials.




Uses:

Artificial ligaments & tendons

Wound dressings

Etc. Darwin's bark spider (Caerostris darwini), an orb-weaver that spins the toughest silk known to science is comparable to 500 megajoules per cubic meter

Spider-Man's webbing is a proportional equivalent of that of a real spider, namely a weaker orb-weaver spider Dragline silks’ tensile strength is comparable to that of high-grade alloy steel

The force needed to stop four New York City subway cars packed with nearly 1,000 people total would be 300,000 newtons

Estimating the toughness of Spider-Man's silk would need to be almost 500 megajoules per cubic meter Primary structure is mainly highly repetitive glycine and alanine blocks

Secondary structure alanine is mainly found in the crystalline domains in beta sheets. Glycine is in the amorphous matrix

Interplay between the hard crystalline segments, and the strained elastic semi-amorphous regions, that gives spider silk its extraordinary properties http://www.livescience.com/27430-spiderman-silk-could-stop-a-train.html

ENGINEERING PROPERTIES OF SPIDER SILK Frank K. Ko1, Sueo Kawabata2, Mari Inoue3, Masako Niwa4, Stephen Fossey5 and John W. Song

http://www.google.com/urlsa=t&rct=j&q=&esrc=s&source=web&cd=5&ved=0CGMQFjAE&url=http%3A%2F%2Fcosmos.ucdavis.edu%2Farchive%2F2008%2Fcluster8%2Fchau_anthony.pdf&ei=1_SHUbSFJoKoigLL4YCoCw&usg=AFQjCNECoSBezxekJjKDFaQnaqE9hGYCZA&sig2=j_m7ip3ZsP4R4VilAlyyNw&bvm=bv.45960087,d.cGE References So yes, Spiderman's silk is indeed realistic!

Clearly spider silk is incredibly strong and elastic which allows for many applications in the real world

The question is how to go about obtaining it in large quantities Is Spider Silk as Strong as Spider Man Makes it Look? Combination of crystalline sections linked by irregular elastic amino acids. It is renounced for being stronger than steel by mass and is surprisingly elastic

Generated interest for an array of applications both medically and in every day life

These properties are a result of both its structure and chemical make up. Silk Duct Spiders naturally secrete fibers for housing, web construction, defense, capturing and detaining prey, egg protection, and mobility known as spider silk

The silk is secreted from glands inside the spiders spinnerets, located on the back of a spiders abdomen. Background
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