Fig. 4.

Possible high throughput mutant construction and analysis. Only minor modifications to the procedures for mutant construction and analysis used in this work are required to greatly increase throughput and compatibility with ultrahigh throughput sequencing methods. (A) Random mutagenesis of portion of gene using mutagenic crossover PCR, error prone PCR, annealing degenerate single-stranded synthetic oligonucleotides, etc.; length of mutant portion of gene limited by sequencing method employed. (B) Construction of growth competition-ready mutant strain library using gap repair cloning. (C) Pooled mutant growth competition. (D) Mutant segment isolation and sequencing preparation using PCR. (E) Chip-based ultrahigh throughput sequencing