Changes made to the Araneus spider silk DNA sequences. (A) The codon frequency is a measure of the abundance of codon sequences in the E. Coli genome relative to each amino acid. The average frequency is the mean of the codon frequencies across the entire sequence of the silk gene. The average codon frequency is shown for the spider (gray) and synthetic (black) genes. Very rare codons (<10 per gene, defined as frequencies <0.13) were entirely eliminated from the sequences. (B) The DNA sequence entropy of the repetitive units is shown for the wild-type spider (gray) and synthetic (black) genes. The repeat units for each silk monomer were manually aligned (Supplementary information) and the sequence entropy is calculated: , where N is the length of the repeat unit and p_i(j) is the probability that base j (A, T, G, C) occurs at position i. The maximum of this function (when all four based are equally represented at each position) is ∼0.6. A lower sequence entropy indicates a higher degree of sequence identity between the repeat units. ADF-3 has extremely repetitive DNA sequences and this repetitiveness is effectively eliminated upon optimization. (C) The amino-acid sequences of the synthetic spider silk genes are shown. Each silk sequence is labelled with a name and the gland in which it is produced. The repetitive regions used in the sequence entropy calculations are in red and green.

Secretion assays are shown for pCASP plasmid, strain variations, and silks. (A) The supernatant contains significant secreted DH protein for the pCASP plasmid (lane 1). Protein secretion is significantly reduced when the chaperone is not co-expressed on the plasmid (-sicP, lane 2) or the N-terminal SptP secretion tag is absent (−Tag, lane 3). (B) A western blot showing that expression of the chaperone SicP does not cause protein leakage. A variant of pCASP was generated lacking the SptP tag (+sicP –sptP); DH protein was expressed in a secretion assay. No protein was detectable in the supernatant until it was concentrated 16 × and exposed for 1 min (data not shown). (C) Secretion of the DH domain from wild-type Salmonella typhimurium SL1344 (WT) is compared with two knockouts. There is little effect when the flagella master regulators are knocked out (ΔflhDC). The accumulation of protein in the supernatant (Sup) is significantly reduced when the prg-org operon (including prgHIDKorgABC) is knocked out (Δprg-org), but can still be detected in the lysate (Lyse). (D) A Coomassie stained gel of concentrated protein (equivalent to 1.5 ml of supernatant) is shown for lysed cells (WT lysis, lane 1), culture supernatant from wild-type Salmonella, (WT Sup, lane 2), and cells secreting the DH domain (DH Sup, lane 3). The DH domain accumulates significantly in the supernatant. The identity of the DH band was confirmed by immunoprecipitation of the protein from supernatant (not shown). The pattern of other secreted proteins in the supernatant matches those reported earlier (Collazo and Galan, 1996). Below the Coomassie gel, a western blot is shown. The periplasmic protein MalE is detectable in the lysate but not in either the wild type or DH supernatants. This indicates that lysis is not significantly contributing to the proteins isolated from the secretion assay. (E) A secretion assay is shown for the synthetic ADF-1, -2, and -3 genes (lanes 2–5). When the N-terminal secretion tag is removed from the sequence (−Tag), no protein is detected in the supernatant (lane 1). The secretion yields with and without the N-terminal tag are determined using quantitative westerns (Supplementary information) and presented in Table II. (F) TEV protease cleaves the SptP secretion tag from the silk proteins. ADF-2 before digestion (lane 1) is reduced in size by 19 kDa when the SptP secretion tag is removed by TEV protease (lane 2).

The dynamics of the sicA promoter and the testing secretion efficiency of tag–chaperone pairs. (A) The time-dependent activation curve of psicA fused to GFP in Salmonella SL1344 (gray circles) and psicA fused to GFP in E. coli DH10B (gray squares) as measured by flow cytometry. Error bars represent ±1 s.d. of three to five independent measurements. Cells are grown in SPI-1 repressing conditions and at T=0 are shifted into SPI-1 inducing media. At 2.5 h the population starts to turn on and by 3 h the majority of cells are in the on state. A western blot for secreted DH protein shows that protein in the supernatant is a function of time. An arrow has been superimposed on the graph indicating the activation of the prgHIDKorgABC operon (encoding the secretion needle). (B) The assay to find the optimal secretion tag/chaperone pair is shown. All known SPI-1 N-terminal tag and chaperone pairs are tested for the secretion of ADF-2 (Table I). Each pair was cloned into the pCASP plasmid under the control of the sicA promoter between the XhoI and HindIII restriction sites. A secretion assay was performed and the western blot of supernatant and lysate samples is shown. The same comparison was made for each remaining silk protein (Supplementary information).