Figure 4

Site-directed saturation mutagenesis using MUPAC. (a) DNA sequence chromatogram obtained by sequencing a plasmid mixture from >10⁴ clones. NNK codons, where K represents G or T were simultaneously introduced to the GFPuv gene at the five specific sites by using MUPAC. CFU for the experiment is indicated in Additional file 1: Table S3. (b) Distribution of nucleotide bases in the five randomized codons. Twenty-four plasmids obtained from individual clones in the saturation mutagenesis experiment were sequenced. The distribution of nucleotides is shown in pie diagrams for each of the randomized base. The nucleotide base A is depicted in green, T in red, G in black, and C in blue. (c) Amino acids appeared at the five sites. Twenty-four plasmids obtained from individual clones in the saturation mutagenesis experiment were sequenced. Translated amino acids at the five sites are shown in one-letter style, and stop codon is denoted by asterisk.