Positive selection and negative screen based on the suppression of Asp112TAG in the chloramphenicol acetyltransferase gene. pBR-TyrRS encoding the mutant MjTyrRS and pAC-tRNA encoding the suppressor tRNA were cotransformed in DH10B cells. (A) Positive selection was carried out in GMML liquid medium with various concentrations of chloramphenicol and p-azido-L-phenylalanine. (B) Negative screen was carried out in GMML liquid medium with various concentrations of chloramphenicol in the absence of p-azido-L-phenylalanine.

After incorporation of multiple copies of plasmid-encoded Methanococcus jannaschii-derived suppressor tRNA, a series of selections based on the suppression of Asp112TAG in the chloramphenicol acetyltransferase gene were carried out. (A) In order to assess the efficiency of the multiple copies of suppressor tRNA, pBR-SD-TyrRS10 encoding the mutant MjTyrRS was cotransformed with pAC-tRNA and pAC-tRNA₄, respectively, in DH10B cells. pAC-tRNA encoded a single suppressor tRNA and pAC-tRNA₄ encoded multiple copies of suppressor tRNA. Positive selection was carried out in GMML liquid medium with 100 μg/mL chloramphenicol and p-azido-L-phenylalanine. (B) pBR-SD-TyrRS4 encoding the mutant MjTyrRS was cotransformed with either pAC-tRNA or pAC-tRNA₄ in DH10B cells. Positive selection was carried out in GMML liquid medium with 100 μg/mL chloramphenicol and p-azido-L-phenylalanine. (C) pBR-SD-TyrRS6 encoding the mutant MjTyrRS was cotransformed with either pAC-tRNA or pAC-tRNA₄ in DH10B cells. Positive selection was carried out in GMML liquid medium with 100 μg/mL chloramphenicol and p-azido-L-phenylalanine. (D) In concordance with the positive selections of A–C, positive selection was carried out in GMML liquid medium with various concentrations of chloramphenicol in the presence of p-azido-L-phenylalanine in order to detect the survivability with chloramphenicol. (E) Negative screen was carried out in GMML liquid medium with various concentrations of chloramphenicol in the absence of p-azido-L-phenylalanine.

Kinetics of uric acid concentration decrease during conversion of uric acid to allantoin by uricase.

After placement of a SD sequence before the start codon for the plasmid-encoded mutant MjTyrRS, positive selection and negative screen were carried out. These selections were based on the suppression of Asp112TAG in the chloramphenicol acetyltransferase gene. pBR-SD-TyrRS encoded the mutant MjTyrRS that was cotransformed with pAC-tRNA in DH10B cells. (A) Positive selection was carried out in GMML liquid medium with various concentrations of chloramphenicol and p-azido-L-phenylalanine. (B) Negative screen was carried out in GMML liquid medium with various concentrations of chloramphenicol in the absence of p-azido-L-phenylalanine.

Western blot analysis of the expression levels of uox-Phe¹⁷⁰-Azp and uox-Phe²⁸¹-Azp under various conditions. An anti-His6 antibody was used to detect the expression levels of the uricase mutants. Cells from 200 μL of the cultures were pelleted and applied to gels for analysis.