This method can help answer key questions in the bioluminescence field. Such as, finding optimal growth conditions for maximal light intensity of bioluminescent bacteria and determining the regulatory mechanism. The main advantage of this technique is the easy setup, which can be used for various strains and organisms, as well as the simple and fast adjustment possibilities.
Following this method can provide insight into lux operon characteristics. It can also be applied to other systems, such as bacterial, environmental, reporting systems, or sensors. To begin, prepare an overnight culture by inoculating 100 milliliters of LB medium with kanamycin, with lux operon transformed E.coli BL21 cells from glycerol stock, or directly from a transformation plate.
Incubate the culture at 37 degrees Celsius, and 120 RPM overnight. The following day, inoculate 800 milliliters of LB kanamycin medium with 8 milliliters of the overnight culture. Incubate the culture at 37 degrees Celsius, and 120 RPM in an incubator shaker, until the cell density reaches an OD600 of 0.6 to 0.8.
Reduce the incubation temperature to 28 degrees Celsius, and induce protein expression by adding 0.1 millimolar IPTG. Then observe the cells until they start to shine. After preparing media according to the text protocol, streak the bacterial bioluminescent strains on artificial sea water medium agar plates, and incubate them at 24 to 30 degrees Celsius overnight.
Prepare an overnight culture by inoculating 100 milliliters of artificial sea water medium with a single colony from the plate. Then incubate the culture at 24 to 30 degrees Celsius and 120 RPM overnight. The following day, inoculate 800 milliliters of artificial sea water medium with 8 milliliters of overnight culture.
Incubate the bacterial cells at 24 to 30 degrees Celsius, and 120 RPM and observe the cells until they begin to shine. Streak the desired bioluminescent bacterial strain, or modified E.coli strain on an agar plate, and incubate the culture at 28 degrees Celsius overnight. 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.
This figure compares OD650 measurements of E.coli BL21 cells, BL21 cells containing an empty pET28a vector, and BL21 cells containing a pET28a vector with the lux-rib operon insert, but without induction. All three reference measurements for light emission show a sigmoidal growth curve with lag, exponential, and stationary growth phases, but no light emission, except for the latter sample, which shows light emission after 300 minutes, due to leakiness of the T7 promoter. Here, the growth curves and light intensities of the lux operon expressed in E.coli, and the bioluminescent bacterial strain, photobacterium mandapamensis 27561, were compared at 28 degrees Celsius in LB, or artificial sea water medium.
The modified E.coli strain and P.mandapamensis grow in artificial sea water medium, as well as LB medium, and both show light emission. But in LB medium, P.Mandapamensis does not emit light at all. Remarkably, the modified E.coli strain shows a higher maximal light intensity than the bioluminescent bacteria.
In this experiment, gene expression of the E.coli based lux-rib operon was measured over 24 hours to analyze its longevity. Light emission lasted for 19 and a half hours, much longer than the bacterial strains, where a gradual decrease was observed, resulting in very low light emission after 10 hours. After its development, this technique paved the way for researchers in the field of bioluminescence to explore lux operons in model organisms, such as E.Coli.
After watching this video, you should have a good understanding of how to work with bioluminescent bacteria.
