A Positive Selection for Nucleoside Kinases in E. coli
A) 2500 CFUs of E. coli BW14012 cells with tat-hsvtk, pelB-hsvtk or dsbA-hsvtk were plated on LB agar plates containing 50 μg/mL streptomycin, 2.5 mM ATP, 1mM IPTG, and the presence (bottom row) or absence (top row) of 10 μg/mL AZT. The plates were incubated for 20 hours at 37°C. B) Western blot using anti-HSV-TK antibodies of periplasmic (“P”) and cytoplasmic (“C”) fractions of cells expressing the indicated proteins. The expect size of HSV-TK is 41 kDa. A control using anti-GroEL antibodies was used to confirm there was no cytoplasmic protein contamination in the periplasmic fractions.