pUC19-lacZα assembly assay.

(A) Two fragments were PCR-amplified from the pUC19 vector to create an efficient screen for DNA assembly capability. The smaller “insert” fragment contained the coding sequence of the lacZα gene starting at position five and some downstream vector sequence. The larger “vector” fragment contained the rest of the plasmid, including the Amp resistance gene (bla) and the origin of replication. The fragments shared 50-bp homology at both ends. (B) Blue colony formation as a function of DNA concentration. Very few white colonies were observed on any of the plates (S3 Table). Small numbers of blue colonies present in the vector-only transformations are indicative of the small amount of contaminating circular template pUC19 used in PCR-mediated linearization of the vector and undigested during DpnI treatment. Insert-to-vector molar ratio was maintained at 5:1, and 25 μl of cells were used, corresponding to ¼ of the recommended volume. (C) Effect of insert-to-vector molar ratio on assembly efficiency. The vector DNA quantity was maintained at 0.5 ng. Error bars indicate standard deviation from two independent sets of experiments.