This study investigates the role of a metal-responsive gene in wild-type bacteria compared to mutant bacteria lacking this gene. The experiment assesses the growth of both types of bacteria in a metal-depleted environment and their ability to extract metal ions from a complex.
Take wild-type bacteria and mutant bacteria that lack a specific metal-responsive gene present in the wild-type.
The bacteria are grown in metal-depleted media to exhaust their internal metal reserves.
In this metal-depleted environment, the metal-responsive gene in wild-type bacteria is activated and expresses a surface receptor, which is absent in mutant bacteria.
Dilute the cultures to ensure uniform cell density.
Take a multiwell plate containing a metal-protein complex.
Add the diluted cultures to the wells.
Incubate the plate inside a plate reader maintained at a controlled temperature with shaking to promote optimal bacterial growth.
Measure absorbance at regular intervals to monitor bacterial growth over time.
In wild-type bacteria, the metal receptor enables the extraction of the metal ion from the complex, supporting its growth.
Mutant bacteria show restricted growth as they fail to extract the metal ion from the complex without the metal receptors.
Add 10 microliters of 10X pre-mix and 90 microliters of CDM to each of three wells for the blank controls.
Once the cultures in the side-arm flask have doubled, add 100 microliters of each culture to an unused well in the microplate, and measure the optical density at 600 nanometers.
After calculating the correct amount of dilution required to bring the cultures to an optical density at 600 nanometers of 0.02, dilute the cultures with CDM in small culture tubes, and add a sufficient volume to dilute the 10X pre-mixes to 1X in the plate.
Then, place the plate in a plate reader for 8 to 12 hours with shaking to obtain optical density at 600 nanometers readings at the desired experimental intervals.
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