Assessing the Protective Role of an Acid-Activated Chaperone in E. coli Under Acid Stress

Take recombinant E. coli cultures sensitive to environmental stress.

The test culture carries a plasmid encoding an acid-activated chaperone, while the control culture carries only the plasmid backbone.

Inoculate single colonies into antibiotic-supplemented media to ensure plasmid retention and incubate to promote bacterial growth.

Dilute the cultures in fresh media containing the antibiotic and an inducer to activate chaperone expression.

Grow the cultures to mid-log phase, then dilute to achieve a specific optical density.

Add acid to lower the pH, inducing acid stress.

In the test culture, acidic conditions activate the chaperone, which stabilizes cellular proteins and prevents their aggregation. In the control culture, the absence of the chaperone causes cellular proteins to denature and aggregate.

Add a base to neutralize the pH and stop the acid stress.

Monitor bacterial growth by measuring optical density over time.

Enhanced growth in the test culture indicates the chaperone’s protective role during acid stress.

Prepare an overnight culture in 50 milliliters of LB with Ampicillin, and cultivate the cells at 200 RPM and 30 degrees Celsius. Dilute overnight cultures 40 fold into 25 milliliters of LB with Ampicillin.

Grow the bacteria in the presence of 0.5% arabinose at 30 degrees Celsius and 200 RPM to an optical density at 600 nanometers, or OD₆₀₀ equal to 1.0, to induce HDEB protein expression. For the pH shift experiments, use LB with Ampicillin and arabinose to dilute the cells to an OD₆₀₀ of 0.5. Adjust to the respective pH values by adding appropriate volumes of five molar hydrochloric acid.

After the indicated time points, neutralize the cultures by addition of the appropriate volumes of five molar sodium hydroxide. Monitor the growth of the neutralized cultures and liquid culture for 12 hours at 30 degrees Celsius using OD measurements.