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Experiments and Protocols



Experimental Protocols



Prepatory Protocols



Heat Shock Transformation

  1. Thaw heat competent cells on ice
  2. Mix 1 - 5 μl of DNA (usually 10 pg - 100 ng) into 50 μL of cells in a microcentrifuge tube, gently mix by flicking
  3. Leave cells on ice for 30 minutes
  4. Heat shock at 42 °C for 30 seconds
  5. Place tubes back on ice for 2 minutes
  6. Add 250 μL of SOC media and 3 µL DAP
  7. Incubate the cells at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate
  8. Warm selection plates to 37°C
  9. Remove 20 μL of cells and plate for a low concentration
  10. Spin cells down for 2 minutes at 8,000 rpm for 2 minutes
  11. Remove most of the supernatant by either decanting or pipetting
  12. Resuspend the cell pellet in the remaining supernatant (80-100 µl) and plate Incubate overnight at 37°C


Electroporation Transformation



  1. Place the following materials on ice: Electroporation cuvettes, (thawed) plasmid DNA, and a conical tube with sterile LB. Label Eppendorf tubes for each transformation.
  2. When everything else is ready, get the electrocompetent cells (50 µL aliquots) out of the freezer and let them thaw on ice.
  3. Add plasmid DNA (1-5 µL) to the cells.
  4. With a pipette set at 50 µL, pipette up and down once to mix.
  5. Then (using the same tip), pipette all 50 µL into an electroporation cuvette.
  6. Electroporate at 1800 V and record the time constant.
  7. Immediately add 1 mL of cold LB to the cuvette.
  8. Decant the LB/cell mixture into a labelled Eppendorf tube.
  9. Recover at 37°C for 1.5 hours.
  10. Centrifuge cells for 1 minute at 15,000 rpm.
  11. Remove most of the supernatant by either decanting or pipetting.
  12. Resuspend the cell pellet in the remaining supernatant (80-100 µl) and plate.
  13. Incubate overnight at 37°C.


Conjugative Baseline Assay



Day 0
  1. Transform E. coli WM6026 with GFP plasmid. Grow overnight on LB agar plate +0.3mM DAP + Amp
Day 1
  1. Pick colonies from the GFP donor strain plate that are expressing GFP
  2. Grow E. coli WM6026 with GFP plasmid overnight, shaking with antibiotic (Amp) and DAP (0.3 mM). Grow in LB. Grow recipient strain E. coli C41 overnight in LB
Day 2
  1. Spin down 3824g 5 min, wash both strains in 1 mL LB +DAP+ (3x)
  2. Combine the 2 suspensions in a 20 mL 1:1 OD ratio in a microfuge tube, pipette to mix
  3. Take OD600 measurements of both cultures (aim for OD of 1.0)
  4. Add 20mL suspension to 50mL conical tube). Cap and place in shaking incubator for 24 hours
  5. Also grow 10mL of each culture in individual 50mL conical tubes, take OD600 measurements at each time intervals, plate recipient sample on Kan in same way as below for as many timepoints as possible
  6. Taking samples: start culture at 3:00 pm, take samples at 4:00 pm, 6:00 pm, 8:00 pm, 10:00 am, 3:00 pm
  7. Take 1.5 mL of the culture and wash with LB (Spin down at 3824g for 5 minutes)
  8. Measure OD600 of 1mL in the spectrophotometer
  9. Section plate into quarters
  10. Perform serial dilution. In quadrant 1, plate 25uL of undiluted culture. Perform 3 serial dilutions, 1:10 each time (undiluted, 1:10, 1:100, 1:1000) . Plate 25uL of the dilution in the appropriate quadrant
  11. Label each plate with the dilution, what time the sample was taken, and plate type
  12. Plate each sample on Kan+Amp, DAP+Kan+Amp
  13. For the controls, plate recipient on DAP+Kan+Amp once, plate recipient on Amp-only once
Day 3
  1. Perform a luminescent screen and record the number of colonies expressing GFP on each plate
  2. Record the total number of colonies on each plate


Synterception Protocols



Lysis Assay and Establishing a Standard Curve



  1. Transform recipient E. coli strain with GFP plasmid (as you would for the baseline assay). Grow in 250mL LB broth overnight at 37°C
  2. Grow untransformed recipient strain in 250 mL LB broth overnight
  3. Grow untransformed recipient strain in 250 mL LB broth overnight
  4. Remove 200 mL broth from each culture. Perform 10 serial dilutions (by a factor of 2), such that each dilution has a final volume of 30mL
  5. Lyse each dilution using a french press
  6. Centrifuge for 20 minutes at 2000xg to pellet cell debris in each sample
  7. For each dilution, remove 1 mL of the supernatant and place in a cuvette. Quantitate GFP fluorescence with spectrophotometer
  8. Remove 1 mL of broth from each culture. Perform 5 serial dilutions (by a factor of 2) and plate on LB agar, incubate overnight
    9. (This is to establish a concentration in CFUs/mL of the original culture, in order to determine how many cells were initially lysed)
  9. Plot the number of cells lysed vs. GFP fluorescence
  10. Perform basic HGT assay. On day 2 before centrifugation, extract 1.5mL of broth from the large culture and spin down in an Eppendorf tube for 20 minutes at 2000xg. Transfer supernatant to cuvette and quantitate fluorescence in supernatant using spectrophotometer. Proceed with assay.


Transformation Assay



Day 1
  1. Grow E. coli overnight, shaking in LB at 37°C
Day 2
  1. Remove 50 mL of culture in a 50mL conical tube and centrifuge 20 minutes at ~13,000xg or max speed
  2. Remove supernatant. Wash with PBS and centrifuge again
  3. Remove supernatant. Resuspend pellet in 10mL LB
  4. Repeat this 7 times to get 7 different tubes.
  5. Add:
    1. Tube 1 : No plasmid (Control)
    2. Tube 2 : Undiluted plasmid (~50ng/µL)
    3. Tube 3 : 1/75 dilution
    4. Tube 4: 1/100 dilution
    5. Tube 5: 1/200 dilution
    6. Tube 6: 1/500 dilution
    7. Tube 7: 1/1000 dilution
  6. Plate 100 uL of each dilution onto an LB plate. Plate 100 uL of each dilution onto an antibiotic plate. Incubate for 8-12 hours
Day 3
  1. Record the total number of colonies on each plate. To calculate the transformation frequency: (Number of Antibiotic CFUs)/(Total CFUs on LB)


Gibson Assembly



  1. Calculate the optimal amounts of DNA for assembly: pmols =(weight in ng)x1000/ (base pairs x 650 daltons). Use the NEB BioCalculator to determine amounts of backbone and insert to add.
  2. Add x μl of backbone to the PCR tube
  3. Add x μl of the insert to the PCR tube
  4. Add x μl of NEB HiFi DNA Assembly Mix to the PCR tube (1:1 ratio of Assembly mix to total DNA)
  5. Incubate the reaction for 15 minutes at 50 ºC


SPLiCE Ligation



  1. Calculate the optimal amounts of DNA for assembly: pmols =(weight in ng)x1000/ (base pairs x 650 daltons). Use the NEB BioCalculator to determine amounts of backbone and insert to add.
  2. Add x μl of backbone to the PCR tube
  3. Add x μl of the insert to the PCR tube
  4. Add 1 μl of SLiCE buffer to the PCR tube
  5. Add 1 μl of SLiCE extract to the PCR tube
  6. Add nuclease free water to get 10 μl reaction
  7. Incubate for 15 minutes at 37 ºC


Colony PCR



  1. Place 50 μl of sterile water in as many PCR tubes as colonies that are being screened.
  2. Pick colonies and place in corresponding water PCR tube, pipette up and down to mix.
  3. Set up a reaction as follows (per tube, so can be scaled up):
    • 5 μl MasterMix
    • 3 μl H2O
    • 0.5 μl forward primer
    • 0.5 μl reverse primer
    • 1 μl of diluted cells
  4. Use NEB Tm Calculator to determine the correct annealing temperature for the primers used in the PCR. Calculate the extension time as 30 seconds per kB.
  5. Run the PCR reaction in the thermocycler