Generation and Culture of Salmonella typhimurium mCherry

To generate S. typhimurium-mCherry, the plasmid pAW83-mCherry was isolated with a miniprep kit (Qiagen) and then transfected into electrocompetent Salmonella enterica serovar typhimurium strain SL1344 through the use of electroporation. Transfected cells were grown on selective LB agar with 100 μg/ml carbenicillin, and a single colony was selected to produce 15% glycerol stocks for −80°C storage as well as confirmation of mCherry expression.

To establish long-lived quantified stocks for infection, the S. typhimurium-mCherry was grown to exponential phase in a large volume of LB broth with 100 μg/ml carbenicillin. The exponential growth was then centrifuged, the pellet was washed once, and then reconstituted in ice cold sterile saline/dextrose (5% dextrose and 0.45% sodium chloride) with 15% glycerol to generate an optical density (OD) value equivalent to 1e9 CFU/ml. It was then split across several small aliquots stored at −80°C, and an aliquot was plated after freeze-thaw to confirm CFU accuracy.