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Figure 5 | Microbial Cell Factories

Figure 5

From: Quantification of the physiochemical constraints on the export of spider silk proteins by Salmonella type III secretion

Figure 5

The construction and testing of many size variants for different silk types. Silk DNA sequence information was obtained from Genbank for multiple silk genes including Latrodectus hesperus Major Ampullate Spidroin 1 (LHF1), Nephila clavipes Flagelliform silk (NCF1), Araneus diadematus Major Ampullate Spidroin 2 (ADF4), and Bombyx mori Heavy Chain Fibroin (BMF1), all of which were optimized and synthesized using chemical gene synthesis. A. Long constructs such as full length LHF1 and the partial clone of NCF1 were further divided using PCR to create libraries of truncation mutants. Mutants include (protein: # amino acids): 8B: 329, 12B: 446, 16B: 568, 20B: 685, 24B: 802, 28B: 921, 32B: 1043, 36B: 1165, 40B: 1286, 1B: 252, 2B: 672. B. A DNA gel showing PCR products for various truncations. Representative LHF1 truncations show a number of size variants up to full length. A PCR product of the ADF3 gene is included to demonstrate the utility across multiple silk sequences. C. Western blots show the detection of silk protein expression and secretion. Each lane is labeled for the concentration of sample loaded and protein identity.

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