Oct 16, 2021
E. coli growth curve assay in BG-11 + sucrose
- 13rd year BS-MS, IISER Pune
- iGEM IISER Pune India 2021
Protocol Citation: sanjana 2021. E. coli growth curve assay in BG-11 + sucrose. protocols.io https://dx.doi.org/10.17504/protocols.io.by5apy2e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: October 16, 2021
Last Modified: October 16, 2021
Protocol Integer ID: 54146
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.
Primary culture
Primary culture
Inoculate 1 colony of E. coli KJK01 in 5 mL of 1X LB
Inoculate 1 colony of E. coli pCSCX-KJK01 in 5 mL of 1X LB with 5 µL Kan50 and 5 µL Amp100
Leave both cultures in a shaker incubator Overnight at 37 °C and 180 rpm
Secondary culture
Secondary culture
The next morning, inoculate 900 µL of 1° culture of KJK01 in 90 mL BG-11 and 10 mL of sucrose. This serves as a control for sucrose consumption.
Expected result
This culture is expected to show negligible/no growth and no butanol production.
Inoculate 900 µL of 1° culture of pCSCX-KJK01 in 90 mL BG-11 and 10 mL of sucrose with 90 µL Kan50 and 90 µL Amp100. This serves as a control for butanol production.
Expected result
This culture is expected to show fastest growth and low butanol production.
Inoculate 900 µL of 1° culture of pCSCX-KJK01 in 90 mL BG-11 and 10 mL of sucrose with 90 µL Kan50, 90 µL Amp100 and 10 µL IPTG (0.1 millimolar (mM) )
Expected result
This culture is expected to show slow growth and maximum butanol production.
Place these three cultures in a shaker incubator at 37 °C and 180 rpm
Preparing samples
Preparing samples
6h 15m
6h 15m
At 06:00:00 intervals, draw 21 mL samples from each culture
6h
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 5000 rpm, 7°C for 00:15:00
15m
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 96:00:00
4d