E. coli growth curve assay in BG-11 + sucrose
sanjana
Abstract
To obtain growth curves of KJK01 and pCSCX-KJK01 in BG-11 + sucrose and to characterize their growth. This also serves to estimate sucrose consumption and butanol production over time.
This can be adapted for any other bacteria as well by giveing proper growth conditions, and using appropriate wavelength to measure the OD and giving proper time intervals based on whether the bacteria is slow growing or fast growing.
Steps
Primary culture
Inoculate 1 colony of E. coli KJK01 in 5mL of 1X LB
Inoculate 1 colony of E. coli pCSCX-KJK01 in 5mL of 1X LB with 5µL Kan50 and 5µL Amp100
Leave both cultures in a shaker incubator at 37°C and 180rpm
Secondary culture
The next morning, inoculate 900µL of 1° culture of KJK01 in 90mL BG-11 and 10mL of sucrose. This serves as a control for sucrose consumption.
Inoculate 900µL of 1° culture of pCSCX-KJK01 in 90mL BG-11 and 10mL of sucrose with 90µL Kan50 and 90µL Amp100. This serves as a control for butanol production.
Inoculate 900µL of 1° culture of pCSCX-KJK01 in 90mL BG-11 and 10mL of sucrose with 90µL Kan50, 90µL Amp100 and 10µL IPTG (0.1millimolar (mM) )
Place these three cultures in a shaker incubator at 37°C and 180rpm
Preparing samples
At 6h 0m 0s intervals, draw 21mL samples from each culture
Measure the OD of 1 sample from each culture at 600 nm and note it down for the growth curve assay
Centrifuge all 6 samples at 5000rpm,7°C,0h 0m 0s for 0h 15m 0s
Decant the solution and store the supernatants at -20°C
Submit 1 sample of each culture for the sucrose assay. This helps determine consumption of sucrose over time.
Submit 1 sample of each culture for NMR analysis. This helps determine butanol production over time.
Continue this procedure for 96h 0m 0s
