The DNA sequence encoding amino acids 1 to 421 of FutA was subjected to error-prone PCR using 0 to 0.3 mM Mn2+ to control the extent of mutation with 0.03 ng of pUC18-FutA as the template. The primers used for this step can be found in table S4. The PCR products were digested and ligated into the pUC18 vector using the HindIII and EcoRI restriction sites, and the ligation mixture was electroporated to E. coli 10G ELITE cells (Lucigen, USA) according to the manufacturer’s protocol. The transformed cells were grown overnight at 37°C in LB medium supplemented with ampicillin (100 mg/ml), and the library plasmid DNA was extracted. Several individual clones from each library were verified by sequencing; this indicated average mutational frequencies of ∼2, ∼4, ∼6, and ∼7 mutations per gene in the 0, 0.1, 0.2, and 0.3 mM Mn2+ libraries, respectively. The plasmid DNA from the four libraries was mixed in a 1:1:1:1 ratio and was then transformed into E. coli JM107* cells for screening.

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