Figure 1.
Schematic of PFunkel mutagenesis using a ssDNA template.
The basic protocol is depicted. For multiple-site mutagenesis, the addition of the polymerase, dNTPs, ligase, DTT, and NAD+ is delayed until after the first annealing step. For comprehensive codon mutagenesis, the ratio of oligo to template is kept low to minimize multiple mutations in a single reaction product. Cycling with occasional spiking of additional mutagenic oligos improves the reaction yield.
Table 1.
Statistics of comprehensive codon mutagenesis library CCM-1.
Figure 2.
Completeness and frequency of codon substitutions observed in 454 sequencing of the comprehensive codon mutagenesis library of TEM-1.
(a) Number of the 63 possible codon substitutions observed and (b) frequency of codon substitutions observed as a function of position in the gene. For each of the 287 codons of TEM-1 the frequency of each of the 63 possible codon substitutions is shown, except for the 3% of the 18,081 codon substitutions that were not observed. The frequency is based on 454 sequencing in which 738,615 codon substitutions were observed in 787,488 reads. The frequency is normalized to the frequency that would occur if all substitutions were evenly distributed among the 18,081 possible substitutions (i.e. frequency = 1.0 means that the substitution was observed 738,615/18,081 = 41 times). The number of codon substitutions observed resulting from sequencing errors is small (∼4% of the 738,615 codon substitutions observed).
Table 2.
Potential adaptive amino acid substitutions in TEM-1 identified from genetic selections for tazobactam resistance codon substitutions.
Figure 3.
Tazobactam resistance of selected alleles.
The increase in ampicillin or piperacillin resistance is reported as the fold increase (over TEM-1) in the minimum inhibitory concentration (MIC) of the antibiotic in the presence of 6 µg/ml tazobactam. MIC assays performed in √2-fold increments of antibiotic concentration. Median MIC values of three replicates were used. Data for all replicates is in Tables S6 and S7.