The overall goal of this methodology is to evaluate a multiple enzymes using a single high-throughput screening system. This method can help answer key questions in the microorganism and bioengineering field about metagenomic screening and enzyme engineering. We have developed a genetic enzyme screening system which is consisting of the MPL mutant responsing phenylene compound and it's downstream GFP reporter.
Once any metagenomic gene shows the enzyme activity of our interest by reacting with the provided substrate, a phenylene compound of the reaction product activate Dmp mutant and trigger GFP reporter expression which can easily be captured by a fluorescence meter. The main advantage of this technique is that unlike conventional screening techniques, this single method is applicable for screening multiple types of different enzymes by using the appropriate substrate. Demonstrating this procedure will be Kil Koang Kwon, a graduate student in our laboratory.
After constructing a metagenomic library in E.Coli with a fosmid vector using a fosmid library production kit, transport the pGESS(E135K)DNA into the metagenomic library cells. Then store the transformed library cells at minus 70 degrees Celsius and 20 microliter aliquots. To remove the false positives, first thaw a metagenomic library cell aliquot on ice.
Then inoculate ten microliters of the thawed cells in two milliliters of lysogenate broth containing ampicillin and chloramphenicol in a 14 milliliter round-bottom tube at 37 degrees Celsius and 200 RPM for four hours. Meanwhile, set the appropriate parameters on the FACS machine for reading the metagenomic library cells in the default FACS software. At the end of the incubation, transfer five microliters of cells into one milliliter of PBS in a five milliliter round-bottom tube.
Then, load the diluted library sample and adjust the event rate to 1000 to 1500 events per second. To generate a log-scaled forward scatter area versus log-scaled side scattered area scatter plot on a Global Worksheet, create a dot-plot and draw a polygon gate around the singlet events containing the bacterial population. To plot a cell count versus log-scaled FITC-area histogram, open a histogram and adjust the FITC voltage such that the peak of the bell-shaped distribution is less than ten to the two of the FITC-area.
Next, open up another dot-plot to create a log-scaled forward scatter area versus log FITC-area plot and set an R2 sorting gate between plus five and minus five percent of the cells from the center of the distribution. When all of the gates have been set, place a collection tube containing 1.2 milliliters of antibiotic supplemented lysogenate broth at the FACS instrument outlet and sort one times ten to the sixth of the gated cells into the broth. At the end of the sort, cap the collection tube and gently vortex the cells.
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 one 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 cellswith 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 five microliters of cells from each sample into individual five milliliter round-bottom tubes containing one 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.
Then place a collection tube containing 0.5 milliliters of lysogenate broth at the FACS instrument outlet and collect one times ten to the fourth of the cells within the R1 and R2 gates. When all of the cells have been isolated, cap the collection tube and gently vortex the cells. Then spread 0.5 milliliters of the collected samples on to two 90 millimeter agar plates containing lysogenate broth supplemented with antibiotics then incubate the plates overnight at 37 degrees Celsius.
In this figure, the demonstrated screening protocol is illustrated including the removal of the false positives by negative sorting, and the positive hit selection using the R1 and R2 sorting gates. The hits can then be evaluated by comparing the sample florescence with and without the substrate. In this representative p-nitrophly acetate mediated screening, the florescence of the substrate-treated cells was higher than that of the control cells, confirming five lipase candidates in this experiment.
Despite the broad distributions of these three cellulose candidates, clear differences in the average FITC intensity of the populations are observed. These four phosphatase candidates also demonstrate high florescence levels compared to the control cells. Further, the alkaline phosphatase activity of orf inserted vectors can be confirmed by flow cytometry with a stronger florescence signal observed for the enzyme hit than for the cells carrying the empty plasmid.
It's important to remember that this technique can be applied to the screening of a broad range of enzymes by choosing a proper substrate and ration concentration. Following this procedure, other methods like next generation sequencing can be performed to answer additional questions about the identification and characterization of the enzyme candidate in a high-throughput manner. After its development, this high-throughput enzyme screening technique paved the way for researchers in the field of enzyme engineering to explore various metagenomic enzymes without needing an enzyme specificity.
After watching this video, you should have a good understanding of how to use the metogenic circuit-based high-throughput screening system with a broad range of enzyme target.
