Computational protocol: Relationships between physical properties and sequence in silkworm silks

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Protocol publication

[…] Silk fibres were positioned on the surface of 1-mm-thick sheet of poly(methyl methacrylate) using double-sided tape on either end and covered in a thin layer of 95% cyanoacrylate and allowed to set overnight. The embedded fibres were sectioned transversely using a RM2265 microtome (Leica Microsystems, Wetzlar, Germany). Fibre cross-sections were visualized using an Olympus BX53 microscope at 500× total magnification and images were taken using Stream Essentials (Olympus, Tokyo, Japan). Cross-sectional surface areas were calculated by tracing the edge contours of the core filaments using ImageJ (NIH, Bethesda, MD, USA). Average values for surface areas were used for A. aliena, R. fugax, and S. jonasii. [...] Amino acid sequences of the fibroins used for the sequence analysis were retrieved from GenBank: Bombyx mori (GenBank accession code AF226688; AAB31861), Samia cynthia ricini (BAQ55621), A. pernyi (AAC32606), A. yamamai (BAJ11925; AB542805), A. assama (KJ862544), Rhodinia fugax (BAG84270), Saturnia japonica (BAH02016), and Actias selene (ADA59934). Additional sequences were obtained from de novo assembled transcriptome data for A. assama and A. selene). Intragenic tandem repeats of saturniid fibroins were aligned using Geneious R9 (Biomatters Ltd., Auckland, NZ) and scored for the frequency of conserved amino acid residues and motifs. The final tandem repeat flanking the C-terminus was not included in the scoring as this segment is typically divergent from the rest of the repetitive regions. [...] Tukey’s HSD (Honestly Significant Difference) test was used in conjunction with one-way analysis of variance (ANOVA) for single step multiple comparisons to analyse the results of the tensile deformation tests, using IBM SPSS Statistics for Macintosh v. 22. […]

Pipeline specifications

Software tools ImageJ, Geneious, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Bombyx mori