Figure 4.

Activity Screen. (A) P99 mutant libraries were constructed by overlap-extension PCR, ligated into an expression vector, and (B) transformed into E. coli and grown on nonselective medium. (C) Plated bacteria was assayed for expression of His-tagged protein by colony blotting and immunodetection. (D) Colonies expressing His-tagged protein were then assayed for antibiotic resistance on agar plates containing varying concentrations of antibiotic, and (E) the P99 variant expressed in each colony was determined by high-throughput sequencing.