Mutagenesis (error-prone PCR and site-directed)

All site-directed mutagenesis experiments were performed following a published protocol ¹ using the E. coli JM109 strain. The randomized screens at N102 of Dronpa and D159 of TagRFP-T were both performed on a wild-type tandem DpTT template using fully degenerate primers (NNN). Error-prone PCR was performed using Taq polymerase and an in-house dNTP mixture. Eight rounds of consecutive PCR were performed (on Dronpa only) using a reverse primer containing the 8-amino acid linker at the 3′ end. This ensured that the linker was not subject to mutation. The mutation rate was approximated through sequencing, and the 3^rd-to-5^th-round PCR products (approximately < 2% point mutation rate) were selected for further cloning. These mutant Dronpa fragments were ligated to a linearized pRSET-B plasmid containing wild-type TagRFP-T using the sites BamHI/KpnI. All of the ligate was transformed into JM109 cells and plated onto LB-Amp agar. The candidate mutants were then screened on the basis of colony fluorescence. Once a candidate was chosen, its fluorescence intensity was confirmed by averaging the fluorescence of many colonies over a larger area on the LB-Amp agar plate.