Figure 3

Cloning strategy for constructing synthetic spider silk genes. (A) Amino acid sequences of designed silk modules were derived from dragline silk proteins ADF-3 and ADF-4 and back translated into nucleotide sequences using the bacterial codon usage. (B) The cloning cassette comprised restriction sites required for module insertion and multimerization. During gene construction a spacer region was replaced by modules and module multimers. The first codon of each module (ggn) determined the "linking" amino acid glycine. (C) Single modules were connected resulting in controlled assembly of synthetic genes. To study different length repeat units, one or two Q modules were combined with one A module to obtain (AQ) or (QAQ). These repeat units were multimerized to generate synthetic genes coding for the repetitive proteins (rep-proteins) (AQ)_n and (QAQ)_n. The repetitive part of ADF-4 is generally composed of a single consensus module termed C, which was multimerized to obtain the rep-protein C_n. Additionally, carboxyl-terminal non-repetitive (NR)-regions from the natural genes were amplified by PCR and optionally linked with the synthetic genes [50].