Experiments

The protocols for the experiments that we used.

Preparing LB + corresponding Plates

  1. Measure corresponding amount of distilled water (usually 1 L when preparing LB for experimental use and 500 mL for preparing plates) into a graduated cylinder and transfer it into an erlenmeyer flask
  2. Add a stir bar into the flask and place it onto a magnetic stirrer
  3. As we have access to LB broth, just weigh out 2.5% of this broth and add into the flask. If no access to this compound, weigh out 1% (w/v) of tryptone and NaCl and 0.5% (w/v) of yeast extract separately and add them into the beaker/flask (For 1 L of LB, that makes 10 g of tryptone, 10 g of NaCl and 5 g of yeast extract)
  4. Wait until the solution is completely dissolved
  5. If preparing LB for agar plates, add 1.5% (w/v) of bacto agar into the flask and swirl it to break up clumps of agar
  6. Cap the flask with a foil and attach a piece of autoclave tape on top of the foil
  7. If preparing LB for experimental use, distribute the solution into 150/250 mL graduated glass bottles (be careful not to add more than 125/200 mL into each as the solution can bump out while being autoclaved)
  8. Put caps into each bottle but do not tighten completely and add autoclave tapes onto them
  9. Autoclave the liquids accordingly (only people with proper autoclave training must use it)
  10. LB media for experimental purposes is ready to use as soon as cooled down
  11. After autoclaving cycle, transfer the flask containing bacto agar into a 50 °C water bath.
  12. Wait until the solution cools down (usually between 20 - 40 minutes)
  13. While waiting, prepare and label corresponding sterile petri plates
  14. Add corresponding antibiotics into the solution by using a micropipette once cooled down (usually 0.1% (v/v) of antibiotic will be added)
  15. Put the flask onto a magnetic stirrer and stir around 1-2 minutes to mix antibiotics with the solution
  16. Aseptically pour down the solution into petri plates (be careful but as fast as possible as the agar will solidify once cooled down)
  17. If necessary, get rid of any bubbles forming by gently touching the bubble with the flame

Transformation by Chemical Competence (Transforming an IGEM Plasmid)

  1. Pipette 5 µL of dH₂O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be red, as the dried DNA has cresol red dye.
  2. Transform all 5 µL of the resuspended DNA into 100 µL of competent E. coli DH5⍺
  3. Incubate the cells on ice for 30 minutes after DNA is added
  4. Heat shock at 42.5 °C for 1 minute
  5. Add 1 ml LB to tube and let cells recover at 37°C with shaking for an hour
  6. Pellet the cells at 14000 RPM for a minute (this is especially crucial when transforming an iGEM Plasmid as the DNA provided is not a lot), discard the supernatant but leave around 100 µL of the supernatant, resuspend the pellet with the leftover supernatant and plate 100 µL of the resuspension onto the corresponding plate
  7. Grow ON at 37 °C
  8. The next day pick up the plates from the incubator and store them in the -4 °C fridge

Transformation by Chemical Competence (Transforming a Miniprepped Plasmid/Gibson - GG Product)

  1. Transform 2 µL of the resuspended DNA into 50 µL competent E. coli strain
  2. Incubate the cells on ice for 30 minutes with DNA added
  3. Heat shock at 42.5 °C for 1 minute
  4. Add 1 ml LB to tube and let cells recover at 37 °C with shaking for an hour
  5. Plate cells on LB + corresponding antibiotics and grow overnight at 37 °C incubator (volume to be plated depends on transformation efficiency)
  6. Another thing to do might be pelleting the cells at max speed for a minute after plating 100 µL of transformed cells, aseptically resuspending the pellet in 100 µL of LB and plating another 100 µL onto another plate)
  7. Grow ON at 37 °C
  8. The next day, pick up the plates from the incubator and store them in the -4 °C fridge

Minipreps to Collect plasmid DNA from the cells (based on Qiagen Miniprep Kit)

  1. Pick a single well-isolated colony aseptically and resuspend in 5 mL of LB + corresponding antibiotics and grow overnight (for ~16 hours) at 37 ˚C with shaking
  2. The next day, transfer 1.5 mL of the solution into a microfuge tube and pellet the cells by centrifuging at max speed for a minute
  3. Get rid of the supernatant and transfer another 1.5 mL of the overnight culture into the microfuge tube
  4. Centrifuge for another 30 seconds and remove the supernatant with a pipette
  5. Resuspend the pellet in 250 µL of Buffer P1 (with RNase already added) (mix well by vortexing or pipetting up and down)
  6. Add 250 µL of Buffer P2 and mix by gentle inversion 4-6 times (by no means should you vortex or pipette up and down this solution) and let it sit for 2-3 minutes
  7. Add 350 µL of Buffer N3 + gentle inversion 4-6 times
  8. Centrifuge for 10 minutes (~13500 RPM)
  9. Label a QIAprep spin column and put it into a 2 mL collection tube
  10. Transfer the supernatant into the spin column (usually around 700 - 800 µL)
  11. Centrifuge at maximum speed for a minute + discard the flow-through + place it back
  12. Add 750 µL of Buffer PE + maximum speed centrifuge + discard the flow-through
  13. Centrifuge for another 1 - 2 minutes to get rid off any remaining material
  14. Transfer the spin column into a labeled microfuge tube
  15. Add either 50 µL of EB solution or dH₂O into the center of the membrane (use the one that works with higher efficiency) + let it sit for 2 - 3 minutes
  16. If the plasmid to be isolated is large, preheat the solution to be added to 50 ˚C and add the heated solution into the center
  17. Centrifuge at max speed for 1 minute + discard the spin column and quantify the DNA by using a NanoDrop
  18. Blank the NanoDrop with 2 µL of Milli-Q water
  19. Load 2 µL of your sample and note the absorption values
  20. Collected plasmid DNA can be stored in eppendorf tubes at -20 ˚C

Preparing glycerol stocks of any E. coli strain

  1. Pick a single well-isolated colony aseptically and resuspend in 5 mL of LB + corresponding antibiotics and grow overnight (for ~16 hours) at 37˚C with shaking (200-250 RPM)
  2. The next day, aseptically add 500 µL of the culture into a glycerol stock tube
  3. Add 500 µL of 50% glycerol into the tube, mix thoroughly by pipetting up and down and store it at -80 ˚C

Preparing an ON culture of any E. coli strain

  1. Wipe down the bench with 70% ethanol
  2. Get a sterile test tube and turn on the Bunsen Burner
  3. Add 5 mL of LB media in the tube aseptically using a plastic pipette
  4. Add the appropriate volume of antibiotics if necessary (NOTE: Use chloramphenicol at a final concentration of 34 µg/mL and kanamycin at 25-50 µg/mL)
  5. Pick a single well-isolated colony aseptically using a toothpick and inoculate into the liquid media
  6. Grow overnight (for ~16 hours) at 37 ˚C with shaking (200-250 RPM)

PCR and Agarose Gel electrophoresis

  1. Resuspend an isolated colony into 20 μL of Milli-Q water
  2. Calculate in advance volumes of all the components required for the PCR reaction
  3. Prepare your master mix by keeping the microfuge tube on ice (add water first and DNA polymerase the last)
  4. If you're using different primers for your PCRs, you can simply create one Master Mix and then distribute the corresponding amount into different microfuge tubes containing each primer pair set
  5. Add the appropriate volume of Master Mix into PCR tubes and add 3.0 μL of the resuspended DNA if using a colony PCR - If you're adding purified DNA instead of a colony PCR please do the calculations and add the appropriate amount of DNA (keep PCR tubes on ice as well)
  6. Make sure to include a NC everytime with no DNA and with the appropriate amount of sterile Milli-Q water
  7. Setup the thermocycler based on the details of your PCR reaction and the Tm of your primers

Setting up an agarose gel

  1. To make 1% agarose gel, measure 0.25 mg agarose in 25 mL of 1X TAE buffer into a flask
  2. Dissolve the agarose into the buffer by heating it up in a microwave (pay attention to the solution as it might bump out once it starts boiling)
  3. Cool down the solution by putting the edges of the flask under the water
  4. Quickly but carefully add 0.5 µg/mL (1.5 µL in this case) of EtBr into the solution in the fume hood and swirl to mix the solution
  5. Pour down the agarose into the gel tray and put the corresponding comp onto it
  6. Let it sit for 15 minutes for the gel to be solidified
  7. Run your samples (mix the dye and DNA on a parafilm)
  8. Run the gel for ~15-20 minutes at 100 Volts and for ~35-40 minutes at 50 Volts (until the yellow band on the ladder disappears)
  9. Get the gel and visualize it in a computer

Primer Resuspension & Dilution

  1. Calculate the amount of Milli-Q water required to create a 100X dilution
  2. Vortex primer stock tubes for 5-10 seconds vigorously
  3. Add the corresponding amount of Milli-Q water and pipette up and down to mix thoroughly
  4. Get some sterile microfuge tubes, label them accordingly, add 10 µL of 100X primer resuspension and 90 µL of sterile Milli-Q water, repeat this process for each primer. These will give us stocks with 10X working concentration
  5. Keep all primer resuspensions + dilutions in an ice box at -20 ºC

Creating Competent Cells (Modified Inoue Method)

  1. Pick a single bacterial colony (2-3 mm in diameter) from a plate that has been incubated for 16-20 hours at 37 ºC. Transfer the colony into 25 mL of LB broth in a 250 mL flask. Incubate the culture for 6-8 hours at 37 ºC with vigorous shaking (250-300 rpm).
  2. At about 6 o'clock in the evening, use this starter culture to inoculate a subculture: the flask receives 50 µL of the incubated culture and 125 µL of LB. Incubate it overnight at 18-22 ºC with moderate shaking.
  3. The following morning, read the OD600 and continue to monitor the OD every 25-30 minutes until it reaches to 0.55
  4. Distribute the culture into 4 different falcon tubes ~30 mL into each. Transfer those tubes to an ice-water bath for 10 minutes.
  5. Harvest the cells by centrifugation at 4000 RPM for 10 minutes at 4 ºC. (cool down the centrifuge to 4 ºC prior to this step)
  6. Pour off the supernatant, resuspend cells by gently pipetting up down in each tube with 10 mL of ice-cold Inoue transformation buffer.
  7. Harvest the cells by centrifugation 4000 RPM for 10 minutes at 4 ºC.
  8. Pour off the supernatant, resuspend the cells in each tube gently in 2.5 mL of ice-cold Inoue transformation buffer
  9. Combine the resuspensions into one falcon tube ~10 mL
  10. Add 0.7 - 0.75 mL of DMSO drop-wise while swirling the culture, ~7% final volume. Mix the bacterial suspension by swirling and then store it in ice for 10 minutes
  11. Working quickly, dispense aliquots of the suspensions into chilled, sterile microfuge tubes - aliquots of 250 - 500 uL
  12. Keep those tubes at -80 ºC for further use
NOTE: Volumes are subject to change based on our decision of how much comp cell to create; yet, keep in mind that each volume to be added will also change in this case

Kirby-Bauer Media Preparation

  1. Add 38 g of Meuller Hinton Agar in 1 liter of distilled water
  2. Add 20% Glucose
  3. Add 33 µl of Methylene blue
  4. Using heated magnetic stir plate - stir contents until dissolved and solution is boiling
  5. Immediately remove once boiling
  6. Autoclave contents on liquid 45 setting (~1h, 15min)
  7. Pour out plates under sterile conditions. Let plates cool 30-40 minutes before use. If not in use, place plates in bag then leave at 4 ºC

Kirby-Bauer Fungi Resuspension & Plating

  1. Re-streak single colony isolate onto new plate - incubate overnight. Must be streaked, not spread to ensure SCI is created
  2. Take 1 colony from sub-culture plate and inoculate it in 0.9% saline (~2ml). Note: 0.9% saline can be made with dH₂O + NaCl -> Autoclave L45 Setting
  3. Vortex at low speed for 5 seconds
  4. Measure optical density at 530 nm. Must be between 0.09-0.12. If higher, dilute by adding more saline. If lower, concentrate by adding more colonies (this is not optimal)
  5. Soak a sterile swab in inoculated saline (~20 sec)
  6. Bring swab to top of tube, using moderate pressure, press the cotton into the side of the tube, rotating the swab around the whole top of the tube (helps prevent excess moisture from going onto the plate)
  7. Using the same swab, streak out the colonies. When streaking, ensure the swab reaches the ends of the plate, and no gaps appear between new swab lines. Swab the whole plate in a downwards direction, then from left to right, then diagonally.
  8. Add Soaked paper discs in 4 regions based on their concentration of Nisin
  9. Incubate overnight
  10. Take a picture of inhibition zones and draw them on top of the plate at 12 hours, 24 hours, 36 hours, and 48 hours. (24 hour mark is crucial, others are for more data, but not absolutely necessary)

Kirby-Bauer Disk Preparation

  1. Either obtain paper disks from a professor, or make your own using paper and a hole puncher
  2. In a small tin-foil covered beaker, autoclave the paper disks
  3. Tape down the tin-foil using autoclave tape
  4. Ensure forceps are sterile - dip in ethanol, then light on fire to remove ethanol - let cool before touching disk
  5. Using forceps, soak 4 paper disks in the 4 different Nisin concentrations and place on the plate in 4 different quadrants. Ensure disks are placed evenly spaced from one another, and each has a reasonable amount of room for the zone of inhibition to form around it on all sides.

Kirby-Bauer Test

Quadrant 1 - Stock nisin Quadrant 2 - 1/10 nisin Quadrant 3 - 1/20 nisin Quadrant 4 - negative control
100 μL nisin 10 μL nisin + 90 μL ddH₂O 5 μL nisin + 95 μL ddH₂O 100 μL ddH₂O
  1. Streak fungi on plates as described above
  2. Immediately place soaked paper disks in their 4 quadrants as described above
  3. Incubate at 37 ℃ overnight (~24 hours) as described above. Additional measurements as described above are optional and to be done only if time permits
  4. When reviewing zones of inhibition, draw the zone on the top of the plate in sharpie
  5. Using a ruler, measure the diameter of the zone
  6. Determine whether the fungi is resistant
  7. Take note of any growth within the zone. No need to culture these further, but could be useful information for future analysis