Bacterial growth in 3000 ug/ml carbenicillin (antibiotic)
Growth/Fitness
Optical Density (OD)
unitless
UV Absorbance Spectroscopy
37°C
None
Assessing bacterial growth on plates containing reporter antibiotic.
Chimeric construct plasmids containing the genes for five Gβ1 variants (MonA, Gβ1-WT, MonB-WT, MonB A45V, and MonB A45Y) were transformed into 50 μl of BacterioMatch two-hybrid system reporter strain compe- tent cells (Stratagene). Cells (80 μl) from each transforma- tion reaction were plated on LB control plates (CK) that contained chloramphenicol (12.5 μg/ml) and kanamycin (50 μg/ml). The function of the control plates was to verify colony numbers in the absence of reporter antibiotic. The same volume of cells (80 μl) was plated on reporter antibiotic plates (CCK) that, in addition to chloramphenicol (12.5 μg/ml) and kanamycin (50 μg/ml), contained carbenicillin (both 750 μg/ml and 1000 μg/ml). All plates were incubated at 37 °C for approximately 20 h.
Assessing bacterial growth in liquid medium containing reporter antibiotic
Plasmids that contained the chimeric construct and the genes for all Gβ1 variants tested were transformed into BacterioMatch two-hybrid system reporter strain compe- tent cells (Stratagene) and plated on control CK plates that contained kanamycin (50 μg/ml), and chloramphenicol (12.5 μg/ml). Colonies were picked from these CK plates and grown in 20 ml of LB liquid medium with kanamycin (50 μg/ml), and chloramphenicol (12.5 μg/ml) overnight at 37 °C. From the overnight cultures, cells were diluted to A600 of 0.1 in 10 ml of fresh LB containing kanamycin (50 μg/ml), and chloramphenicol (12.5 μg/ml). The cultures were then incubated for 15 min at 37 °C and the A600 was measured again to verify that all cultures were the same density. Carbenicillin (3000 μg/ml) was then added, and the cultures were incubated for a total of 4 h, at which time the A600 was measured to ascertain the extent of growth.