Team:Guelph/Experiments




Experiments

LB Media

  1. 20 g LB broth powder
  2. Mix up to 1L water
  3. Autoclave

LB Agar

  1. 20 g LB broth powder
  2. 15 g agar
  3. Mix up to 1L water
  4. Autoclave

Preparing and Running a 1% Agarose Gel

  1. Obtain a beaker and dispense 45 mL of TAE buffer.
  2. Weigh and pour 0.45 g of agarose powder into the beaker containing the TAE buffer (1% concentration of agarose powder to TAE).
  3. Microwave the solution until all agarose is dissolved. Ensure that a Kimwipe is placed in the opening, covering the top of the beaker.
  4. Let gel solution cool to lukewarm temperature (hot to the touch but can be held in your hand without burning you).
  5. Pipette 2.25 μL of redsafe into the prepared 45 mL liquid (1 μL / 20mL).
  6. Slightly swirl the mixture (avoid creating bubbles) and then pour the mixture in the taped up gel casting tray.
  7. Wait for the liquid to solidify to gel state.
  8. Remove the tape and load the gel casting tray into the gel cassette (ensure adequate TAE buffer is present in cassette to submerge the gel).
  9. Place 2 μL of 6x loading dye onto parafilm.
  10. Add and mix 10 μL of DNA (and ladder) for each sample of loading dye.
  11. Pipette up each sample and place them into their respective well. Record the order of samples.
  12. Run the gel at 92 V until the dye front is ¾ of the way through the gel.

Single Tube Transformation Protocol

  1. Label sterile 1.5 mL tubes
    1. One for each transformation
    2. Pre-chill tubes on ice
  2. Thaw competent cells on ice
    1. This may take 10-15 minutes
  3. Pipette 50 μL of competent cells into the sterile 1.5 mL tubes per transformation
  4. Pipette 20 μL of DNA per 50 μL of competent cells
  5. Mix cells gently by pipetting up and down
  6. Incubate cells and DNA in 1.5 mL tubes on ice for 30 minutes
  7. Heat shock cells at 42°C for 30 seconds
    1. The bottom of the tubes should be below the water line
    2. Make sure the time is exactly 30 seconds
  8. Incubate cells on ice for 5 minutes
  9. Add 950 μL of SOC or LB media to each transformation
  10. Incubate cells at 37°C for 1 hour and shaking at 250 rpm
  11. Pipette all of the contents of the tube onto a petri plate
  12. Spread the liquid using a sterile spreader
  13. Leave plates cracked open beside a flame until dry
  14. Incubate transformations overnight at 37°C

Preparing MIC

  1. Grow the bacterial strains to be tested (E. coli DH5a and BL21) on LB agar plates without antibiotics and incubate at 37°C for 24 hours. These plates should be a streak to isolating type plate.
  2. Inoculate 5 mL of LB broth with a colony from the plates and incubate at 37°C for 24 hours. In the morning replace 1 mL with fresh LB media
  3. Prepare stock solution of antibiotic
  4. Mix stock antibiotic and LB broth to a final volume of 2 mL
  5. Add 200 μL of this to each well in the first column of the plate. This will be the highest concentration of antibiotic used.
  6. Add 100 μL of just LB to the rest of the wells.
  7. Take 100 μL from the wells in the first column and add it to the second column. Continue until one column before the last is reached making sure to discard the tips between each move.
  8. For the last column, discard 100 μL so each well only has 100 μL of media left
  9. Leave the last column alone as this will be the positive control (no antibiotic)
  10. Dilute the overnight broth cultures to an OD of 0.1 using fresh LB with the formula:
    1. (1/OD)xFinal Volume = Amount to of culture to use in μL
  11. Inoculate each well with 100 μL of LB+Culture bringing the total well volume to 200 μL
  12. The negative controls were filled with 100 μL of blank LB
  13. Incubate at 37°C and shaking at 900 rpm

Restriction enzyme double digestion using NEB time-saver enzymes

Incubate at 37 C for at least 2 hours. After the digestion is done, inactivate it by heating it to 65 C for 20 min (for EcoRI and PacI; changes with different enzymes).








Ligation Protocol









  1. Gently mix reaction tubes
  2. Incubate ligations
  3. 10 minutes at room temperature OR
  4. Overnight at 16°C
  5. Heat inactivate the enzymes at 65°C for 10 minutes
  6. Keep on ice until ready for cell transformation
Appendix: Calculating amount of Insert DNA
  1. Decide how much DNA of the vector is to be used in the reaction in ng
  2. Typically use 50 ng of DNA for a 20 μL reaction volume but pick an amount that is reasonable
  3. Can also look at how much of the DNA will have to be added to the 20 μL reaction volume in order to keep it to a lower amount
  4. To calculate the amount of insert needed, use the formula:
Amount of insert (ng) = [insert length (bp) / vector length (bp)] x amount of vector (ng) The amount given by the above formula is the amount of insert needed for a 1:1 ligation ratio. To get other ligation ratios, simply multiply this number by the first number in the ligation ratio required (ex. 3:1 = amount of insert (ng) x 3, 10:1 = amount of insert (ng) x 10)

Miniprep Protocol

  1. Resuspend the pelleted cells in 250 µL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be completely resuspended by vortexing or pipetting up and down until no cell clumps remain. Note: Ensure RNase A has been added to the Resuspension Solution
  2. Add 250 µL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Note: Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
  3. Add 350 µL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. Note: It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should become cloudy.
  4. Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
  5. Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate. Note: Close the bag with GeneJET Spin Columns tightly after each use!
  6. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube. Note: Do not add bleach to the flow-through.
  7. Add 500 µL of the Wash Solution (diluted with ethanol prior to first use as described on p.3) to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
  8. Repeat the wash procedure (step 8) using 500 µL of the Wash Solution.
  9. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
  10. Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 µL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min. Note. An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane. Discard the column and store the purified plasmid DNA at -20°C.

GeneJET Protein purification protocol adapted from ThermoFisher

  1. Add a 1:1 volume of Binding Buffer to completed PCR mixture (e.g. for every 100 µL of reaction mixture, add 100 µL of Binding Buffer). Mix thoroughly. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.
  2. For DNA ≤500 bp Optional: if the DNA fragment is ≤500 bp, add a 1:2 volume of 100% isopropanol (e.g., 100 µL of isopropanol should be added to 100 µL of PCR mixture combined with 100 µL of Binding Buffer). Mix thoroughly. Note. If PCR mixture contains primer-dimers, purification without isopropanol is recommended. However, the yield of the target DNA fragment will be lower.
  3. Transfer up to 800 µL of the solution from step 1 (or optional step 2) to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through. Notes. If the total volume exceeds 800 µL, the solution can be added to the column in stages. After the addition of 800 µL of solution, centrifuge the column for 30-60 s and discard flowthrough. Repeat until the entire solution has been added to the column membrane. Close the bag with GeneJET Purification Columns tightly after each use!
  4. Add 700 µL of Wash Buffer (diluted with the ethanol as described on p. 3) to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through and place the purification column back into the collection tube.
  5. Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove any residual wash buffer. Note. This step is essential as the presence of residual ethanol in the DNA sample may inhibit subsequent reactions.
  6. Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube (not included). Add 50 µL of Elution Buffer to the centre of the GeneJET purification column membrane and centrifuge for 1 min. Note For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 µL does not significantly reduce the DNA yield. However, elution volumes less than 10 µL are not recommended. If DNA fragment is >10 kb, prewarm Elution Buffer to 65 °C before applying to column. If the elution volume is 10 µL and DNA amount is ≥5 µg, incubate column for 1 min at room temperature before centrifugation.
  7. Discard the GeneJET purification column and store the purified DNA at -20 °C.

ThermoFisher Gel Extraction Protocol

  1. Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 mL tube and weigh. Record the weight of the gel slice. Note. If the purified fragment will be used for cloning reactions, avoid damaging the DNA through UV light exposure. Minimize UV exposure to a few seconds or keep the gel slice on a glass or plastic plate during UV illumination.
  2. Add 1:1 volume of Binding Buffer to the gel slice (volume: weight) (e.g., add 100 µL of Binding Buffer for every 100 mg of agarose gel). Note. For gels with an agarose content greater than 2%, add 2:1 volumes of Binding Buffer to the gel slice.
  3. Incubate the gel mixture at 50-60 °C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved. Vortex the gel mixture briefly before loading on the column. Check the colour of the solution. A yellow colour indicates an optimal pH for DNA binding. If the colour of the solution is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.2 solution and mix. The colour of the mix will become yellow.
  4. For ≤500 bp and >10 kb DNA fragments Optional: use this step only when DNA fragment is ≤500 bp or >10 kb long. If the DNA fragment is ≤500 bp, add 1 gel volume of 100% isopropanol to the solubilized gel solution (e.g. 100 µL of isopropanol should be added to 100 mg gel slice solubilized in 100 µL of Binding Buffer). Mix thoroughly. If the DNA fragment is >10 kb, add 1 gel volume of water to the solubilized gel solution (e.g. 100 µL of water should be added to 100 mg gel slice solubilized in 100 µL of Binding Buffer). Mix thoroughly.
  5. Transfer up to 800 µL of the solubilized gel solution (from step 3 or 4) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube. Note: If the total volume exceeds 800 µL, the solution can be added to the column in stages. After each application, centrifuge the column for 30-60 s and discard the flow-through after each spin. Repeat until the entire volume has been applied to the column membrane. Do not exceed 1 g of total agarose gel per column. Close the bag with GeneJET Purification Columns tightly after each use!
  6. Optional: use this additional binding step only if the purified DNA will be used for sequencing. Add 100 µL of Binding Buffer to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
  7. Add 700 µL of Wash Buffer (diluted with ethanol as described on p. 3) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
  8. Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove residual wash buffer. Note. This step is essential to avoid residual ethanol in the purified DNA solution. The presence of ethanol in the DNA sample may inhibit downstream enzymatic reactions.
  9. Transfer the GeneJET purification column into a clean 1.5 mL microcentrifuge tube (not included). Add 50 µL of Elution Buffer to the centre of the purification column membrane. Centrifuge for 1 min. Note. For low DNA amounts, the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 µL does not significantly reduce the DNA yield. However, elution volumes less than 10 µL are not recommended. If DNA fragment is >10 kb, prewarm Elution Buffer to 65 °C before applying to column. If the elution volume is 10 µL and DNA amount is ≤ 5 µg, incubate column for 1 min at room temperature before centrifugation.
  10. Discard the GeneJET purification column and store the purified DNA at -20 °C.

Expression Tests

To get our results we conducted multiple types of expression tests ranging from broth, microbroth and agar assays. Here you can find generally how we conducted these assays.

For our expression test, we used a solution of ampicillin [100 μg/mL], IPTG [1 mM] (if working with Violacein), tryptophan [100 μg/mL] (if working with Violacein) and tetracycline [varied from 1, 10, 50, 100 and 267 ng/mL], all in LB media.

An example of this type of expression test was a test tube method based on techniques used by Liljeruhm et al. to constitutively express amilCP (Liljeruhm et al., 2018).

We made 1 mL cultures in 1.5 mL Eppendorf tubes. The Eppendorf tubes contained 998 mL of LB broth with ampicillin (100 μg/mL) and tetracycline (50 ng/mL) and 2 μL of culture.

Using their method, we tested for the production of pigments in the presence of tetracycline using the following protocol:

Broth

Macrobroth

Day 1:

  1. Create 5 mL LB+ampicillin overnight cultures from glycerol stock.


Day 2:

  1. Dilute the overnight cultures to OD 0.1 in 50 mL of LB in test tubes.
  2. Add ampicillin to a final concentration of 100 μg/mL.
  3. Add tetracycline to a final concentration of 0.1 μg/mL.
    1. If testing with any violacein genes, add IPTG to a final concentration of 1 mM.
    2. Vortex to mix and incubate at desired temperature (4°C, 16°C, 25°C, or 37°C).
  4. Remove tubes after 12 hours and visualize in ambient light.
  5. If color is faint or not visible, cultures can be placed at 2 – 8 °C for up to 7 days.



Microbroth

In our microbroth expression tests, the plates were set up exactly as we set up the MIC assays, however the media is supplemented with IPTG and Amp at their working concentrations.

The plates were set up and incubated either:
i) 37°C, shaking at 900 rpm.
ii) 4°C, 16°C, 25°C, or 37°C

After 24 hours of growth the E. coli was quantified by its recorded OD 600nm and the AmilCP produced was measured at a wavelength of 588 nm. If no colour is visible it was then incubated in the fridge for 4 days and had its pigment production at 588nm, normalized to the 24 hour cell density.

This is done because much growth should not have occurred beyond the 24 hour read as cell division is slowed but the pigment can accumulate beyond what original did after 24 hours.


Agar Media For our plate expression tests, PCR confirmed bacteria were spread on LB agar plates containing ampicillin, IPTG, tryptophan and tetracycline. The following test was performed:

Streak Plate

1. From an overnight or glycerol stock, streak colonies to isolation onto the solid agar.
2. Incubate at desired temperature

Spread Plate

Day 1:
1. From an overnight or glycerol stock, make an overnight in LB-Amp broth. Incubate at 37°C shaking at 250 rpm.

Day 2:
1. Dilute to OD 0.1
2. Pipette 200 μL on a VioSensor Agar Plate
3. Spread using glass beads for an even distribution all over the plate
4. Incubate at 37°C.

Disc Diffusion

Same as the spread plate assay, however before incubating the plate, place the desired drug disc in the centre of the plate.



A variation of the streak and spread plate test was performed to investigate the expression of prodeoxyviolacein in response to IPTG. Colonies of VioABE were spread on LB agar plates with concentrations of IPTG ranging from 0.1 mM to 1mM.

Agar plates were made as follows (per 125mL):

2.5g LB powder, 1.875g agar, 125mL water

1. Make a stock solution of IPTG by dissolving 0.1192 g IPTG in 2mL ddH­2O for a final concentration of 250 mM.
2. Add stocks as follows:
1mM at 500μL; 0.075mM at 375μL; 0.5mM at 250μL; 0.25mM at 125μL; 0.1mM at 50μL
3. Streak colonies from previous plates on to the new plates.






Liljeruhm, J., Funk, S. K., Tietscher, S., Edlund, A. D., Jamal, S., Wistrand-Yuen, P., … Wistrand-Yuen Erik & Forster Anthony. (2018). Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology. J Biol Eng, 12(8). Retrieved from https://jbioleng.biomedcentral.com/articles/10.1186/s13036-018-0100

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University of Guelph iGEM 2019