Detection of Target Enzyme Expression in Genetically Modified Bacterial Cells

Begin with tubes containing genetically modified bacterial cells. Each bacterium carries high-copy fosmids that encode various enzymes.

Incubate the tubes with shaking to facilitate the expression of various intracellular enzymes.

Add a substrate specific to the target enzyme to the test tube and add an equal volume of buffer to the control tube.

Incubate with shaking. The substrate enters the cells, where the target enzymes cleave it, generating a fluorescent product.

Load the control tube into a pre-calibrated flow cytometer.

As each cell passes through a laser beam, it scatters light. Generate a scatter plot to identify single cells, which scatter less light than clumps.

Exclude the cell clumps, and plot the fluorescence signal of the single cells to establish baseline fluorescence.

Place the substrate-treated tube in the flow cytometer to measure the fluorescence signal.

An increased fluorescence signal in substrate-treated cells compared to the control cells confirms the target enzyme expression.

To screen the metagenomic enzymes of interest, incubate the sorted cells at 37 degrees Celsius and 200 RPM until the OD₆₀₀ reaches 0.5. Then add 1 microliter of copy induction solution to ampl