Team:UCLA/Notebook/Recombinant Expression/2 June 2015

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  • Spider Silk Genetics
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• MEETING NOTES
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• Experimental Design Brainstorm

• • Tamura-GFP Hydrogel assay
    • Negative control - sfGFP without Tammy.
    • Experimental protein - Tamura
    • Assay for binding potential to doped silk solution.
      • Used stripping buffers to assay for agitation. (detergents, alkalinity, ionic strength, pH).
    • Differing variables: Concentration of Dope, concentration of Tamura in hydrogel.
    • Temperature based gradient on hydrogel stability.
    • Assay for flourescence using microplane reading, distribution using fluorescence intensity (read into measuring this quality).

Characterization of Tamura -

• • SDS-PAGe gels, Western Blot to assay degradation.
  • CD, IR Spectrum, Fluorescence Intensity of purified protein and compare that with protein concentration - relative to that metric (ug).
  • Cry - EM for silk Tamura fibers - make sure that we don't mess with the fibers.
  • Gel meshes and pore size using EM.

• • Tamura-ABD Assays
    • Incubate in albumin containing solution for set time, measure the rate of release of albumin within the hydrogel. Hydrogel with ABD-Tam, and ABD out.
  • CD, IR, before and post vortex of the solution.

• • Flourescence following washes of Tamura.

• • SElective binding to silk sequence regions.

• • Expectation of 100mg per 1L of cell culture.

• • • 1ng minimum per well for measureflourescnece.

• • • • Tamura - 00.5% of total silk, 2%,

0.5% is 25ug per well, 1% 50 ug per well 2% - 100 ug per well.

• • • • Net STEP: Make hydrogels in PBS tomorrow.