This article describes a method for screening genetically modified bacterial cells to identify target enzyme expression using flow cytometry. The process involves incubating cells with specific substrates and analyzing fluorescence signals to confirm enzyme activity.
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 amplify the intra-cellular fosmid copy number.
After three hours at 37 degrees Celsius and 250 RPM combine 0.5 milliliters of the cells with the appropriate substrate in a 14 milliliter round-bottom tube to a 100 micromolar final concentration. Then incubate the control and experimental samples at 37 degrees Celsius and 200 RPM for another three hours.
While the samples are shaking, set the FACS machine parameters as just demonstrated. Then, add 5 microliters of cells from each sample into individual 5 milliliter round-bottom tubes containing 1 milliliter of PBS. Next, load the sample cells and set the event rate to 1000 to 3000 events per second.
Create a log-scaled forward scatter area versus log-scaled side scatter area scatter plot, and adjust the R1 scatter gate to encompass the singlet event bacterial cells. Then create a log-scaled forward scatter versus log-scaled FITC-area scatter plot and set an R2 sorting gate so that less than 0.1% of the negative cells are detected. Now, load the sample tube and readjust the event rate to 1000 to 3000 events per second, as necessary.
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