Detection of Target Enzyme Expression in Genetically Modified Bacterial Cells

Abstract

Source: Kim, H. et. al., Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System. J. Vis. Exp. (2016)

This video demonstrates a genetic screening method to detect enzyme expression using metagenomic fosmid libraries and chromogenic substrates. Flow cytometry identifies and isolates single cells producing colored products, enabling high-throughput selection of active enzyme-expressing cells.

Protocol

1. Preparing the Metagenomic Library with a genetic enzyme screening system for the detection of p-nitrophenol (pNP) (pNP-GESS)
   1. Construct a metagenomic library in Escherichia coli with a fosmid vector using a fosmid library production kit.
   2. Aliquot 100 µl of the library for storage at −70 °C, which is a source of metagenomic library cells.
      NOTE: The optical density of a sample measured at a wavelength of 600 nm (OD₆₀₀) of this library stock is approximately 100.
   3. Thaw 100 µl of the stock metagenomic library on ice and inoculate in a 500 ml flask containing 50 ml Luria-Bertani (LB) and 12.5 µg/ml chloramphenicol, followed by 37 °C incubation for 2 hr.
   4. Harvest the cells in a 50 ml conical tube by centrifugation at 1,000 x g for 20 min at 4 °C.
   5. Resuspend the pellet quickly in 50 ml ice-cold distilled water (DW) and centrifuge at 1,000 x g for 20 min at 4 °C again.
   6. Resuspend the pellet in 50 µl ice-cold DW with 10% (v/v) glycerol. Use 50 µl of this cell aliquot for electroporation.
   7. Place the mixture of electrocompetent cells 50 µl and pGESS(E135K) DNA (100ng) in an ice-cold electroporation cuvette and electroporate (1.8 kV/cm, 25 µF) the mixture.
   8. Quickly add 1 ml super optimal broth with catabolite repression (SOC) medium and resuspend the cells gently.
   9. Transfer the cells into a 14 ml round-bottom tube using a pipette and incubate at 37 °C for 1 hr.
   10. Spread 500 µl of the recovered cells on an LB 20 x 20 cm square plate containing 12.5 µg/ml chloramphenicol and 50 µg/ml ampicillin. Incubate the plate at 30 °C for 12 hr.
   11. Scrape the colonies using a cell scraper and collect cells into a 50 ml conical tube using ice-cold cell storage media.
   12. Centrifuge at 1,000 x g for 20 min at 4 °C. Resuspend the pellet in 10 ml ice-cold cell storage media to reach an OD₆₀₀ of 100.
   13. Aliquot 20 µl of the cells for storage at −70 °C.
       
2. Removing False Positives from the Metagenomic Library
   1. Thaw the stock metagenomic library cells (from Step 1.13) containing metagenomic DNA fosmid and pGESS(E135K) on ice.
   2. Inoculate 10 µl of the cells in 2 ml LB containing 50 µg/ml ampicillin and 12.5 µg/ml chloramphenicol in a 14-ml round-bottom tube. Incubate at 37 °C with shaking at 200 rpm for 4 hr.
   3. Meanwhile, turn on the fluorescence-activated cell sorter (FACS) machine and open the default FACS software. Use the following settings: nozzle tip diameter, 70 µm; Forward Scatter Area (FSC-A) sensitivity, 300 V-logarithmic amplification; Side Scatter Area (SSC-A) sensitivity, 350 V-logarithmic amplification; fluorescein isothiocyanate area (FITC-A) sensitivity, 450 V-logarithmic amplification; threshold parameter, FSC-A value 5.
      NOTE: Typical settings are given for FACS device mentioned in the Table of Materials. Adjust the settings for other FACS devices.
   4. Dilute the metagenomic library cells from Step 2.2 by adding 5 µl of the sample to a 5-ml round-bottom tube containing 1 ml phosphate-buffered saline (PBS).
   5. Place the diluted library sample onto the loading port of the FACS instrument and click on Load button on the Acquisition Dashboard of the FACS software.
   6. Adjust the event rate to 1,000 - 1,500 events/sec by clicking and controlling the Flow Rate buttons on the dashboard.
   7. Create log log-scaled FSC-A vs. log-scaled SSC-A scatter plot on a global worksheet by clicking on the Dot Plot button in the toolbar. Adjust the scatter gate R1 to encompass the singlet events (bacterial population) using the Polygon Gate button in the toolbar.
   8. Plot a histogram with cell count vs. log-scaled FITC-A on the worksheet by clicking on the Histogram button. Then, adjust the FITC voltage from the Cytometer Settings tab on the Inspector Control window such that the peak of the bell-shaped distribution is less than 10² of FITC-A.
   9. Create a log-scaled FSC-A vs. the log FITC-A plot by clicking on the Dot Plot button on the global worksheet. Set a sorting gate R2 on the plot using the Polygon Gate button on the toolbar so that the gate is located between +5% and -5 % cells from the center of the distribution (a total of 10% cells around the peak of the bell-shaped curve).
   10. Place a collection tube containing 1.2 ml LB containing 50 µg/ml ampicillin and 12.5 µg/ml chloramphenicol at the outlet of the FACS instrument and sort out 10⁶ cells satisfying both the R1 and R2 gates.
   11. Remove the collection tube, cap, and gently vortex.
       
3. Metagenomic Enzyme Screening
   1. Incubate the sorted cells (from Step 2.11) at 37 °C with shaking at 200 rpm until the OD₆₀₀ reaches 0.5.
   2. Add 1 μl copy induction solution to amplify the intracellular fosmid copy number. Incubate the cells for an additional 3 hr at 37 °C with shaking at 250 rpm.
   3. To prepare cells for sorting, add 0.5 ml of the cultured cells and an appropriate substrate (p-nitrophenyl acetate, p-nitrophenyl-β-D-cellobioside, or phenyl phosphate) into a 14-ml round-bottom tube at a final concentration of 100 µM.
      1. For a control sample, add 0.5 ml of the cultured cells from Step 3.2 into a 14-ml round-bottom tube. Add the same volume of PBS as the substrate volume in Step 3.3.
   4. Incubate the two samples at 37 °C with shaking at 200 rpm for 3 hr.
   5. Meanwhile, prepare the FACS machine with the same configurations as Step 2.3.
   6. Add 5 µl of the cells (for sorting) and control cells to two 5 ml round-bottom tubes containing 1 ml PBS, respectively.
   7. Place the tube containing control cells onto the loading port of the FACS and click on Load button on Acquisition Dashboard of the FACS software. Adjust the event Rate to 1,000 - 3,000 events/sec by controlling Flow Rate buttons on the dashboard.
   8. Create log log-scaled FSC-A vs. log-scaled SSC-A scatter plot by clicking on the Dot Plot button on the global worksheet of the software, and adjust the scatter gate R1 to encompass the singlet events (bacterial population) using the Polygon Gate button on the toolbar.
   9. Create log log-scaled FSC-A vs. log-scaled FITC-A scatter plot by clicking on the Dot Plot button on the worksheet, and set a sorting gate R2 on the plot using the Polygon Gate button on the toolbar so that less than 0.1 % of negative cells are detected within the R2 gate.
   10. Replace the control sample tube with the sorting sample tube, and adjust the event rate to 1,000 - 3,000 events/sec by controlling the Flow Rate buttons on the dashboard.
   11. Place a collection tube containing 0.5 ml LB at the outlet of the FACS instrument and sort out 10⁴ cells satisfying both the R1 and R2 gates.
       NOTE: The sorting criterion can range from the top 0.1% to 5% of FITC-A in the R2 gate. In this protocol, the top 1% of cells were collected as positives.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CopyControl	Epicentre	CCFOS110	Fosmid library production kit
CopyControl induction solution	Epicentre	CCIS125	Fosmid copy induction solution
EPI300	Epicentre	EC300110	Electrocompetent cell
pCC1FOS	Epicentre	CCFOS110	Fosmid vector
Gene pulser Mxcell	Bio-Rad		Electroporation cuvette and electroporate system
FACSAria III	Becton Dickinson		Flow Cytometry (FACS machine)
AZ100M	Nikon		Microscope
Ultraslim	Maestrogen		LED illuminator
50-ml conical tube	BD Falcon
14-ml round-bottom tube	BD Falcon
5-ml round-bottom tube	BD Falcon
p-nitrophenyl phosphate	Sigma-Aldrich	N7653	Substrate
p-nitrophenyl β-D-cellobioside	Sigma-Aldrich	N5759	Substrate
p-nitrophenyl butylate	Sigma-Aldrich	N9876	Substrate
Luria-Bertani (LB)	BD Difco	244620	Tryptone 10 g/L, yeast extract 5 g/L, sodium chloride 10 g/L
Super optimal broth (SOB)	BD Difco	244310	Tryptone 20 g/L, yeast extract 5 g/L, sodium chloride 0.5 g/L, magnesium sulfate 2.4 g/L, potassium chloride 186 mg/L
Super optimal broth with catabolite repression (SOC)		SOB, 0.4 % glucose
2x yeast extract tryptone (2xYT)	BD Difco	244020	Pancreatic digest of casein 16 g/L, yeast extract 10 g/L, yeast extract 5 g/L
Cell storage media			2x YT broth, 15% glycerol, 2% glucose
pGESS(E135K)			A DNA vector containing dmpR, egfp genes with their appropriate promoters, RBS, and terminator.See the reference 5 in the manuscript for more details.
Chloramphenicol	Sigma	C0378
Ampicillin	Sigma	A9518
BD FACSDiva	Becton Dickinson		Flow Cytometry Software Version 7.0
PBS	Gibco	70011-044	0.8% NaCl, 0.02% KCl, 0.0144% disodium phosphate, 0.024% monopotassium phosphate, pH 7.4