FIG. 2.
Construction of random libraries. Random libraries for positions 69 and 238 and the combination of positions 69 and 238 in SME-1 were created by a PCR-based approach. (A) Primers were designed to randomize the targeted codon (NNS) and to introduce restriction sites SacI and BamHI at the ends of the PCR products for cloning into the pTP123 vector. (B) The first PCR consisted of the use of one internal mutagenic primer and one outside cloning primer (SME-Sac or SME-BamHI). (C) The PCR products from the first set of reactions were mixed together and amplified by using only the outside cloning primers to regain the full-length blaSME gene with the target position randomized. (D) This PCR product was digested with SacI and BamHI and cloned into the pTP123 vector. MCS, multicloning site.