[Feedback 1] A single colony of both the wild-type and the mutant were inoculated in separate flasks containing 50ml LB broth with 100ug/mL ampicillin. The flasks were placed in 225 rpm shaker at 37°C overnight. The culture was diluted to 0.5L and transferred to the incubator and grown further until the mid-log phase of 0.67 in the range (A600 = 0.6-0.9) was achieved. Protein expression was induced by adding 1mM Isopropyl β-D- 1-thiogalactopyranoside (IPTG). The cultures were incubated in a shaker at 22°C for 4hours.The bacterial cells were harvested by centrifugation at 4°C. The media paste was discarded, and the bacterial pellets frozen at -80°C overnight. Protein extraction was done using the B- PER® Bacterial Protein Extraction Reagent kit as follows: The pellets were resuspended in 16mL of B-PER Reagent containing 20 mM Tris HCl (pH 7.5) with added lysozyme, DNase 1 and EDTA-free protease inhibitors. The mixture was incubated at room temperature for 15 minutes which was followed by centrifugation at 4000 x g for 1 hour to obtain the whole- bacterial cells extract. Our GST-tagged x11FLUc wild-type and x11FLuc Arg262Gln were purified using the Pierce® GST Spin Purification Kit according to the manufacturer’s guidelines and were eluted with 3mL wash buffer (125mM Tris, 150mM sodium chloride, pH 8.0) containing 1 volume of glutathione.