This method uses co cultivation of fluorescently labeled bacillus sutilis to monitor the rapid clonal expansion and elimination of beneficial and detrimental mutations in a bacterial cell population over time. First, prepare the pre cultures of the bacteria for the competition experiment. Mix equal amounts of the fluorescently labeled strains and co cultivate the bacteria under different nutritional conditions.
Then collect the samples at different time points, dilute appropriately and propagate the cells on agar plates. Now visualize and count the surviving strains by stereo fluorescence microscopy. Ultimately, results can determine the percentage of the surviving bacteria with respect to the whole number of counted colonies.
Unlike monitoring intra species competition using different antibiotic re cassettes for strain labeling. In this method, fluorescently labeled competitor strains are almost completely isotonic and capable of brewing on the same AGA plates. The pre procedure will be presented by Lorena, who is a PhD student in my lab inoculate the four milliliters of LB medium with one microliter of bacteria from minus 80 degrees Celsius, cryo stocks, and grow the pre cultures overnight at 28 degrees Celsius and 220 RPM dilute 0.1 milliliters of culture into 0.9 milliliters of LB medium in a 1.5 milliliter vete, and determine the optical density.
Then to obtain mixed cell populations for the competition experiments, inoculate 100 milliliter shake flasks containing 20 milliliters of CGLC and CGLC. Minimal medium in a one to one ratio, transfer 10 milliliters of the cultures to 15 milliliter plastic tubes. Harvest the cells by centrifugation for 10 minutes at 4, 000 times gravity and discard the supernatant.
Then resuspend the cells in 0.5 milliliters of fresh LB medium, and transfer the cells to a sterile 1.5 milliliter reaction cup add 0.5 milliliters of 50%sterile glycerol. Mix the suspension by rigorous vortexing and store the samples in a minus 80 degrees Celsius freezer. Next culture, the remaining bacteria in the shake flasks.
Keep the shake flasks in the dark to prevent photobleaching of the flora force sample 0.1 milliliters of the cultures after seven hours and 24 hours of growth measure and note the optical density of one to 10 dilution of the samples make cryo stocks of the samples to store at minus 80 degrees Celsius. After collecting all the samples, thaw the cryo stocks and dilute the cells in a 0.9%saline solution for each strain plate. 0.1 milliliters of the 10 to the negative fifth dilution on an SP medium agar plate, and distribute the cells using a sterile glass pipette.
Incubate the plates overnight at 37 degrees Celsius in the dark until single colonies have appeared with a black pen. Divide the bottom of the agar plate in four parts for a better orientation while counting the survivors under the stereo fluorescence microscope. Now, place the plate upside down under the microscope and bring the colonies into focus using the cold light source.
Once the colonies are in focus, switch to the appropriate filter set for CFP Flora four. To visualize the surviving cells of the CFP labeled strain, count the survivors of this strain by labeling the colonies with a pen. And note the number.
Remove the labels with ethanol. Then switch to the YFP filter set. To visualize the surviving cells of the YFP labeled strain.
Count the survivors again by labeling the colonies with a pen. And note the number. Open the pictures from one spot that were taken with the CFP filter set and the YFP filter set.
Optimize the contrast and the brightness to reduce any background fluorescence from the media. Go to one of the pictures and select all. Copy the picture and paste it onto the other picture.
Merge the pictures by selecting in the layers menu the option lighter color. In this experiment, a cell population consisting of two BCI strains were labeled with the Fluor CFP and YFP to evaluate the effect of different nitrogen sources on growth by plating appropriate dilution on agar plates. The clonal composition of a cell population over time can be precisely determined simply by counting the yellow and blue fluorescent colonies.
Glutamate dehydrogenase enzyme activity strongly affects the fitness of otus depending on the availability of exogenous glutamate. In the absence of exogenous glutamate, high level GDH activity is disadvantageous for the bacteria as the enzymes rock G and good B degrade glutamate that is needed in anabolism. By contrast, synthesis of two functional gdhs is advantageous for the bacteria when exogenous glutamate is available because in addition to G glucose glutamate is used as a carbon source.
Similar results were obtained in a die switch experiment. Again, bacteria equipped with high level GDH activity were out competed by cells with reduced GDH activity in growth media lacking glutamate. By contrast, bacteria synthesizing only one active GDH were eliminated from the culture.
When the medium was supplemented with glutamate, clearly the initial composition of the mixed cell population remained almost constant over time. Irrespective of Fluor taken together. The usage of Flora Force is a powerful tool for monitoring intra species competition in a bacterial cell population Once the competitor strengths were generated.
This technique can be done in five days if it is performed properly.
