Team:Leiden/Experiments

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Protocols

On this page you can find a list of protocols we used and optimized during our research, each detailing how to carry out experiments the way we performed them. Simply click on a protocol to read its steps. For easy navigation, you can use our side menu to scroll to the protocol you are looking for.

Pouring Agar plates
  1. Add LB-agar to dH2O in the prescribed concentration
  2. Autoclave the obtained LB-agar solution
  3. Let the solution cool down to 60°C and add antibiotics where necessary
  4. Pour the LB-agar in empty Petri dishes until it covers the bottom
  5. Leave the Petri dishes parially open (asceptically) until the agar is completely solidified (approximately 5 minutes)

    Final concentration of antibiotics:
  • Ampicillin: 100 μg/mL
  • Chloramphenicol: 25 μg/mL
  • Kanamycin: 50 μg/mL
  • Tetracyclin: 10 μg/mL
Competent E. coli (DH5α)
  1. Grow E. coli (DH5α) in LB medium at 37°C overnight
  2. Transfer a small fraction of the overnight culture into fresh LB and grow untill the culture reaches an OD600 of about 0.3-0.4
  3. Pellet the cells at 4000 rpm for 10 minutes
  4. Resuspend the cells in TSS buffer
  5. Make aliquots of a desired volume and store at -80°C


TSS buffer is composed of 10% (w/v) polyethylene glycol, 5% (v/v) dimethyl sulfoxide (DMSO) and 50 mM Mg2+ set at pH 6.5.

Competent Bacillus subtilis (strain 168) and subsequent transformation
  1. Make an overnight liquid culture of B. subtilis, grown at 37°C
  2. Transfer the bacteria to SM1 medium (37°C) and incubate at 37°C until an OD600 of 0.5 to 0.7 is reached (4 to 5 hours)
  3. Add an equal volume of SM2 medium (37°C) and incubate for another 90 minutes
  4. Transfer the suspension to a microcentrifuge tube and add 1 μL of ligated construct solution
  5. Incubate at 37°C for 30 minutes under shaking conditions (200 rpm)
  6. Add 60 μL LB medium and incubate for another 30 minutes before plating on appropriate plates


SM1 medium is made with 15 mL ST-base, 150 μL Tryptophane solution (10 mg/mL), 37.5 μL MgSO4 (0.3 M), 300 μL 50x YECA and, 150 μL 50% glucose

SM2 medium is made with 15 mL ST-base, 150 μL Tryptophane solution (10 mg/mL), 150 μL MgSO4 (0.3 M), 150 μL 50x YECA, 150 μL 50% glucose and, 75 μL CaCl2

Genomic DNA isolation E. coli
Using GenElute™ Bacterial Genomic DNA Kit
  1. Pellet an overnight culture of E. coli (5 minutes @ 13 000 rpm) and discard the supernatant
  2. Resuspend the pellet thoroughly in 180 μL lysis solution and 20 μL RNase
  3. Incubate this solution for 2 minutes at room temperature
  4. Add 20 μL of Proteinase K solution and incubate another 30 minutes at 55°C
  5. Add 200 μL of Lysis Solution C and vortex
  6. After 10 minutes of incubation at 55°C, add 200 μL of ethanol (95-100%) to the lysate and mix thoroughly
  7. Transfer the contents into a binding column and spin at ≥6500 ×g for 1 minute
  8. Discard the eluate
  9. Wash the colum with washing solution, spin down and discard the eluate
  10. Repeat the washing step with the same washing solution, this time supplemented with ethanol
  11. To elute the DNA, add 200 μL Elution solution and incubate for 5 minutes at room temperature
  12. Collect the DNA by spinning down the sample at ≥6500 ×g for 1 minute
Plasmid DNA isolation E. coli
Using GenElute™ Plasmid miniprep Kit
  1. Pellet an overnight culture of E. coli (5 minutes @ 13 000 rpm) and discard the supernatant
  2. Resuspend the pellet in 200 μL Resuspension solution
  3. Add 200 μL Lysis solution and invert gently to mix (Do not vortex!)
  4. Incubate this solution for 5 minutes at room temperature
  5. Add 350 μL of Neutralization solution and invert to mix
  6. Pellet the debris (10 minutes @ max-speed)
  7. Meanwhile, wash the binding column with 500 μL Column preparation solution (1 minute @ max-speed) and discard the eluate
  8. Transfer the cleared lysate (supernatant from step 6) into a binding column and spin down at ≥12000 ×g for 1 minute
  9. Discard the eluate
  10. Wash the colum with 750 μL washing solution, spin down and discard the eluate
  11. Spin the column 1 minute to dry the column and then transfer the column to a new collection tube
  12. To elute the DNA, add 100 μL Elution solution and spin down the sample at max-speed for 1 minute
PCR on genomic DNA
  1. Prepare the PCR reaction as is indicated below:
    2. Component Volume (final concentration)
    Taq polymerase buffer (10x) 5 μL
    Taq polymerase 1 μL
    dNTPs 4 μL (200 μM total)
    Forward primer 0.5 μL (0.5 μL)
    Reverse primer 0.5 μL (0.5 μL)
    DMSO 1.25 μL
    MilliQ 36.75 μL
    Total 49 μL
  2. Add 1 μL of genomic DNA (approximately 200 ng) to the reaction mix
  3. Put the sample(s) in a PCR machine and run the following protocol:
    4. Step 1 (1x) 2 min @ 94°C
    Step 2 (30x) 30 sec @ 94°C
    30 sec @ 50°C
    30 sec @ 72°C
    Step 3 (1x) 5 min @ 72°C
    hold @ 12°C
  4. Check the PCR products by performing gel electrophoresis


Tip: Don't forget to make a sacrifice to the PCR gods in order for it to succeed

Single BioBrick Assembly
  1. Restrict the desired BioBrick part with EcoRI and PstI in a suitable buffer for 1 hour at 37°C

    Below you can find the composition of restriction solution that we used:
    2. Compound Volume
    Restriction enzymes 1 μL each
    Cutsmart buffer 2 μL
    BioBrick DNA 5 μL
    MilliQ 11 μL
  2. Restrict the destination plasmid with EcoRI and PstI in a suitable buffer for 1 hour at 37°C

    Below you can find the composition of restriction solution that we used:
    3. Compound Volume
    Restriction enzymes 1 μL each
    Cutsmart buffer 3.5 μL
    Backbone plasmid DNA 3 μL
    MilliQ 11.5 μL
  3. Deactivate the restriction enzymes at 80°C for 20 minutes
  4. Ligate the cut BioBrick and destination plasmid with T4 ligase for 1 hour at room temperature

    Below you can find the composition of ligation solution that we used:
    5. Compound Volume
    BioBrick part 12 μL
    Plasmid backbone 5 μL
    T4 Ligase buffer 2 μL
    T4 Ligase 1 μL
  5. Transform E. coli with the obtained mixture according to the transformation protocol
Dual BioBrick Assembly
  1. Restrict the desired upstream BioBrick part with EcoRI and SpeI in a suitable buffer for 1 hour at 37°C

    Below you can find the composition of restriction solution that we used:
    2. Compound Volume
    Restriction enzymes 1 μL each
    Cutsmart buffer 2 μL
    BioBrick DNA 5 μL
    MilliQ 11 μL
  2. In the same manner, restrict the desired downstream BioBrick part with XbaI and PstI in a suitable buffer for 1 hour at 37°C
  3. Restrict the destination plasmid with EcoRI and PstI in a suitable buffer for 1 hour at 37°C

    Below you can find the composition of restriction solution that we used:
    4. Compound Volume
    Restriction enzymes 1 μL each
    Cutsmart buffer 3.5 μL
    Backbone plasmid DNA 3 μL
    MilliQ 11.5 μL
  4. Deactivate the restriction enzymes at 80°C for 20 minutes
  5. Ligate the cut BioBricks and destination plasmid with T4 ligase for 1 hour at room temperature

    Below you can find the composition of ligation solution that we used:
    6. Compound Volume
    BioBrick parts 5 μL each
    Plasmid backbone 3 μL
    T4 Ligase buffer 2 μL
    T4 Ligase 1 μL
    MilliQ 4 μL
  6. Transform E. coli with the obtained mixture according to the transformation protocol
DNA purification E. coli: Quick and Dirty
  1. Transfer a single colony of transformants into liquid LB medium with appropriate antibiotic and incubate overnight at 37°C
  2. Pellet the cells (5 minutes @ 13 000 rpm) and discard the supernatant
  3. Resuspend the cells in 100 μL STET buffer
  4. Incubate the solution at room temperature for 5 minutes
  5. Add 5 μL lysozyme solution (10 mg/mL)
  6. Incubate 1 minute at 95 to 100°C
  7. Centrifuge the sample (13 000 rpm for 10 minutes) and continue with the supernatant
  8. Add 105 μL cold isopropanol (-20°C) and vortex slightly
  9. Centrifuge the sample (13 000 rpm for 10 minutes) and discard the supernatant
  10. Add 400 μL ethanol (70%) to the pellet and vortex shortly
  11. Centrifuge the sample at 13 000 rpm for 5 minutes
  12. Discard the supernatant and dry the pellet at 65°C for 10 minutes
  13. Resuspend pellet in 50 μL TE buffer containing 1 μL RNase
  14. Incubate at 65°C for 20 minutes and vortex
  15. The obtained solution contains the desired DNA and can be used for gel electrophoresis to check whether a transformation was successfull


STET buffer is composed of 8% sucrose, 0.5% Triton X-100, 50 mM Tris-HCl and 50 mM EDTA (pH 8.0)

TE buffer is composed of 10 mM TRIS and 1 mM EDTA (pH 8.0)

DNA purification B. subtilis: Quick and Dirty
  1. Grow an overnight culture of B. subtilis
  2. Pellet the bacteria (5 minutes @ 13 000 rpm) and discard the supernatant
  3. Resuspend the bacteria in 100 μL TEG buffer
  4. Add lysozyme solution and incubate for 10 minutes at 37°C
  5. Add NaOH/SDS solution and mix until it clarifies
  6. Add NaAC (3 M) to precipitate the cell components
  7. Centrifuge solution at 13 000 rpm for 7 minutes and continue with the supernatant
  8. Add isopropanol (-20°C) and mix the solution
  9. Pellet the sample (5 minutes @ 13 000 rpm) and discard the supernatant
  10. Add 400 μL ethanol (70%)
  11. Pellet the samples again and discard the supernatant
  12. Dry the pellet for 10 minutes at 65°C
  13. Resuspend the pellet in 50 μL TE buffer supplemented with 1 μL RNase and incubate at 65°C for 20 minutes


TEG buffer is composed of 25% (w/v) sucrose, 50 mM EDTA and, 50 mM Tris-HCl (pH 8.0)

NaOH/SDS solution is composed of 0.2 M NaOH and 1% (vol/vol) SDS in MilliQ

Measure DNA concentration (NanoDrop)
  1. Initiate the dsDNA program of the NanoDrop
  2. Clean the sensor and blank with 1 μL sterile MilliQ
  3. Load 1 μL sample and measure the DNA concentration


Note: The A260/280 ratio should be larger than 1.8 and the A260/230 should be larger than 2.0

Gel electrophoresis

Note: This protocol uses Ethidium Bromide, which is a carcinogenic compound. Handle all infected materials with a glove.

  1. Heat a 1% agarose TAE buffer solution to dissolve the agarose
  2. Add Ethidium Bromide to a final concentration of 0.5 μL/mL and transfer the mixture to a gel electrophoresis mold
  3. Allow the gel to solidify
  4. Place the gel into an electrophoresis device filled with TAE buffer and load the desired samples and appropriate DNA ladder(s)
  5. Run the gel at 100 V for 20 to 60 minutes
  6. Transfer the gel to a gel imager and visualize the DNA
Transformation E. coli
  1. Thaw competent E. coli cells on ice
  2. Add 5 μL of ligated construct solution to roughly 50 μL cells and mix carefully by pipetting up and down
  3. Incubate the sample on ice for 30 to 60 minutes
  4. Incubation the sample at 42°C for 90 seconds (heat shock) before returning it to ice again
  5. Keep cells on ice for 5 minutes
  6. Add 1 mL LB medium to the sample and grow at 37°C for about an hour under shaking conditions (200 rpm)
  7. Plate the obtained solution on LB agar plates with appropriate antibiotic(s) and incubate overnight
Long-term storage of bacteria
  1. Grow an overnight culture of the desired bacterial strain at the appropriate conditions
  2. Add twice the volume of 20% glycerol solution and mix thoroughly
  3. Store the bacteria at -20°C to -80°C


Tip: Make sure to label the vials extensively to prevent mistakes later on

Preparation for flow cytometry
  1. Make an overnight culture of the strain(s) to be tested
  2. Dilute the bacteria 50 to 200 times in LB medium (aiming for an OD600 of 0.1 approx.)
  3. Transfer 100 μL culture to each well of a U-shaped 96-well plate
  4. Add 100 μL of stressor solution to each well
  5. Incubate the plate in a microplate incubator shaker at 37°C, 600 rpm for 2 to 4 hours
    Keep bacteria on ice for the subsequent steps
  6. Centrifuge the 96-well plate at 2000 ×g for 5 minutes
  7. Discard the supernatant and resuspend the cells in ice-cold PBS buffer (-4°C)
  8. Centrifuge again and repeat the washing step with PBS
  9. Analyze samples by flow cytometry
Disk diffusion
  1. Inoculate an indicator strain in LB medium and incubate at 37°C untill an OD600 of 0.1 is reached
  2. Distribute the liquid culture uniformly over an Mueller-Hinton agar plate with a sterile cotton swab
  3. Carefully place felt disks on the agar plate and pipette 35 μL of antibiotic-LB solution into the disk
  4. Incubate at 37°C for 24-72 hours


Note: Do not place the petri dish upside down to prevent the disk from falling off

Overlay assay with Streptomyces
  1. Prepare ISP 2 medium agar plates
  2. Make 6 spots (evenly distributed across the plate) of 1 μL glycerol-stock of Streptomyces (e.g. S. coelicolor and S. Griseus in different dilutions)
  3. Incubate the Streptomyces spots for 48 hours at 30°C
  4. Meanwhile, grow an indicator strain at 37°C untill an OD600 between 0.4 and 0.6 is reached
  5. Make a solution of 2% (v/v) indicator strain in soft agar
  6. Pour approximately 5 to 10 mL of the soft agar mixture evently on top of the Streptomyces spots
  7. Incubate overnight at 37°C


ISP 2 medium agar plates is composed of 0.4% (w/v) yeast extract, 1% (w/v) malt extract, 0.4% (w/v) dextrose and 2% (w/v) agar in distilled water

Soft agar is a 1% agarose in LB solution (half the agar concentration than LB agar)

Freeze drying E. coli
  1. Make an overnight culture of the desired E. coli strain
  2. Plate 100 μL of that culture on a petri dish with LB agar
  3. Incubate overnight at 37°C
  4. Add 4 mL of a 1% glycerol solution to the plates and scrape the bacteria loose
  5. Prepare plastics tube for freeze drying by making a hole in the lid to prevent pressure build up in the freeze dryer
  6. Transfer the obtained suspension to the plastic tube
  7. Place the tubes in a freeze dryer container and let the freeze dryer run for at least 24 hours
  8. Carefully remove the tubes and store the bacteria at room temperature
Confocal microscopy
  1. Grow overnight culture of the desired strains
  2. Dilute to an OD600 of 0.1
  3. Add stressors to the desired concentration (if applicable)
  4. Incubate further for 2 to 3 hours
  5. Place a droplet of the culture on a microscopy slide and view under the microscope

Our confocal microscopy pictures were taken with a Zeiss LSM 5 Exciter-Axiolmager M1 confocal microscope with a Plan-Aprochromat objective (63x/1.4 Oil DIC) and Zeiss Zen 2009 software. For GFP measurements, cells were excited at 488 nm and measured using an emission filter of 505-600 nm. E. coli cells with mCherry were excited at 575 nm and emission at 610 nm.

Lethality screening of natural compounds
  1. Prepare stocks of soluble compounds in MilliQ. If using natural products, soak the compounds in milliQ for 24 hours at 60°C. The table below outlines the concentrations used of the products in our experiments.
    Product Concentration (g/mL)
    Garlic (extract) 1.10
    Curcuma (extract) 0.20
    Onion (extract) 1.00
    Honey 0.80
    Red pepper (extract) 1.47
    Ginger (extract) 0.31
    Cinnamon (extract) 0.26
    Caffeine 0.04
    Oregano (extract) 0.20
    Thyme (extract) 0.20
    Liquorice (extract) 0.21
    Ginger pre-packaged (extract) 0.60
    Wasabi 0.25
    Horseradish (extract) 0.52
    Ascorbic acid 112
    Cacao (extract) 167
    Black pepper (extract) 0.30
    Cannabis (extract) 25
  2. Add 100 µL of the extract or stock and 100 µL E. coli culture with an OD600 of 0.1 to a 96 wells plate. A negative control of 100 µL milliQ must be included in the measurement
  3. Incubate plate in a microplate incubator shaker at 37 °C at 600 rpm
  4. Take OD600 measurements every 30 minutes until the OD of the negative control reaches 0.5
  5. Determine relative growth compared to the negative control
MIC determination
  1. Prepare stocks of serial dilutions in antibiotics in LB
  2. Add 100 µL antibiotic stock solution and 100 µL E. coli culture with an OD of 0.1 to a 96 wells plate
  3. Incubate plate in a microplate incubator shaker at 37 °C at 600 rpm
  4. Take OD600 measurements every 30 min until the OD of the negative control reaches 0.5
  5. Determine the minimal concentration at which growth is inhibited
NotebookClick here to visit our Notebook to see which experiments we did when and how they turned out.
PartsClick here to visit our Parts page and see which BioBricks we have constructed and characterized.
SafetyClick here to visit our Safety page and learn about everything concerning safety in our project.