This article describes a method for inducing bioluminescence in bacterial colonies transformed with a plasmid. The process involves measuring cell density and light intensity to correlate growth with luminescence.
Take bacterial colonies transformed with a plasmid containing an inducible gene.
Pick a single colony to inoculate fresh liquid media.
Incubate with shaking for oxygenation and homogeneous bacterial growth.
Use a diluted culture sample to measure absorbance and estimate cell density.
Take a black-walled, glass-bottom well plate to prevent light cross-talk between samples, ensuring accurate luminescence quantification.
Add the bacterial culture with the desired cell concentration into the wells.
Add an antibiotic to maintain plasmid retention.
Add an inducer to initiate gene expression.
The gene encodes an enzyme that reacts with a specific bacterial protein to produce light, giving the bacteria bioluminescent properties.
Place a lid on the plate to prevent evaporation before placing it in a temperature-controlled plate reader with constant shaking mode.
At regular intervals, record absorbance to measure cell number and light intensity to monitor bioluminescence. Correlate cell density with bioluminescence.
Inoculate 3 milliliters of medium with a single colony from the overnight plate, and incubate the cells at 28 degrees Celsius, and 180 RPM for approximately one to two hours.
Measure the cell density of a one-to-ten dilution of the liquid culture at 650 nanometers. Then calculate the ratio and volume for one milliliter of culture with an OD650 of 0.05. Pipette the calculated volume of culture and medium into a 24 welled black walled plate with a glass bottom.
To ensure that the pET28a vector containing the entire lux operon does not get lost, and that is expressed in the modified E.coli strain, add one microliter of kanamycin, and one microliter of IPTG to the samples. Then place a lid on the plate, to avoid evaporation during the measurements. Finally, use a plate reader set to 28 degrees Celsius and a script found in the text protocol, to take absorbance and bioluminescence measurements every 10 minutes, with shaking in between data points.
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