Experiments

iGEM is Following a Pipeline

This section explains the different laboratory protocols followed by the wetlab team.

On our Experiments page, you’ll find all the protocols used throughout our project. Each protocol outlines the step-by-step processes we followed to achieve our results. The accompanying image illustrates the sequence of steps taken during our experiments.

Figure 1. C. acnes competent cells protocol. Scheme made with BioRender.



For the plasmid digestion protocol add in an Eppendorf tube:

  • 1 µg of DNA
  • 5 µL of 10X rCutSmart Buffer
  • 1 µL of SacI-HF
  • Nuclease-free Water up to 50 µL

It must then be incubated at 37°C for 5–15 minutes. After this, the plasmid must go through a clean up by following the PCR purification protocol to obtain the digested plasmid.


The Gibson Assembly method is essential for creating our plasmid constructs. Whether it is for storage or the definitive backbone (pJet or pEnd, respectively), the exact same protocol was followed. To proceed with the Gibson Assembly, introduce the following components in a 0.2 ml Eppendorf tube, in the order listed:

  • 50 ng of template (empty backbones).
  • 3X molar ratio of the part being introduced, with respect to the template used in each case.
  • The same amount of Gibson Mix as the sum of the last two.

The 0.2 ml Eppendorf tube will be then placed into the thermocycler for 1 hour, at 50 ºC.

The Gibson mix contains three key components: T5 exonuclease, DNA polymerase, and DNA ligase. T5 exonuclease creates 3′ overhangs by digesting 5′ ends of the DNA, DNA polymerase fills in any gaps between annealed fragments, and DNA ligase seals the nicks to form a continuous DNA molecule. These enzymes work together in an optimized buffer that maintains the proper conditions for the reaction. For its correct functioning, it is very important to work at low temperatures when manipulating this mix.

Note: It is important to note that this protocol is highly specific to the lab we worked in, as it relies on the prefix and suffix sequences flanking all of our parts, as well as the exact cleavage sites in the backbones.


To set up a DNA PCR or Colony PCR, use the following components in a 0.2 ml Eppendorf tube, in the order listed:

  • 19 µL of H₂O if DNA PCR; 21 µL of H₂O if Colony PCR.
  • 25 µL of 2x Phanta Master Mix.
  • 2 µL of Primer 1.
  • 2 µL of Primer 2.
  • Template DNA (2 µL in case of DNA PCR).

Note: The primers used depend on the specific DNA fragment you want to amplify.

Figure 2. Thermocycler machine used.


To perform the PCR, we must put the Eppendorf PCR tubes in the thermocycler. The specific settings depend on what you are performing the PCR on but here is a table with the most common settings:

Step Temperature Time Number of cycles
Initial Denaturation 94 °C 2 min ---
Denaturation 94 °C 15 sec 30 - 35
Primer Annealing 65 °C 20 sec 30 - 35
Extension 72 °C 1 min/kb 30 - 35
Final Extension 72 °C 5 min ---
Resting temperature 4 °C --- ---

After performing the PCR, you can analyze the results following the agarose gel protocol.


To prepare agarose gels, we used a tray that allowed the creation of four, twenty-well gels simultaneously. The measurements for one gel are as follows:

  • 150 mL of TBE buffer.
  • 1.5 g of agarose.
  • 4.5 µL of Green Safe dye.
  1. First, weigh out 1.5 g of agarose to make a 1% agarose gel. Add the agarose to an Erlenmeyer flask.
  2. Add 150 mL of TBE buffer to the flask and gently mix the contents.
  3. Heat the mixture in a microwave for 45-60 seconds, mixing it until the agarose is fully dissolved.
  4. Allow the solution to cool slightly, then add the Green Safe dye and mix thoroughly.
  5. Pour the solution into the gel tray, ensuring the well combs are in place.
  6. Allow the gel to solidify for about 20 minutes.
Figure 3. Agarose Gel machinery used.



PCR purification is typically done after PCR amplification to prepare the product for sequencing. Follow these steps:

  1. Load 25 µL of the PCR mixture into a PCR purification column.
  2. Add 125 µL of PB buffer and centrifuge at 13,000 rpm for 1 minute.
  3. Discard the flow-through.
  4. Add 750 µL of PE buffer and centrifuge again at 13,000 rpm for 1 minute.
  5. Discard the flow-through.
  6. Centrifuge at 13,000 rpm for 2 minutes to remove any extra buffer.
  7. Transfer the PCR purification column into a new Eppendorf tube.
  8. Add 20 µL of water, wait for 1 minute, then centrifuge at 13,000 rpm for 1 minute.
The purified PCR product will be in the Eppendorf tube.

For the sequencing:

  • In case of sequencing a PCR product, 50-250 ng. If it is a MiniPrep, 150-500 ng.
  • 2.5 µL of desired primer.
  • 2.5 µL of H₂O.
Figure 4. Sequencing samples.



For E.coli transformation:

  1. Gently mix the thawed competent cells by tapping the tube. Do not vortex.
  2. Add 1-5 µL of plasmid DNA (10-100 ng) to the competent cells.
  3. Gently flick the tube to mix the DNA with the cells. Do not vortex.
  4. Incubate the cells with the DNA on ice for 20-30 minutes. This allows the DNA to associate with the cell membrane.
  5. Transfer the tubes to a 45°C Thermoblock for 45-60 seconds.
  6. Quickly return the tubes to ice and incubate for another 2-5 minutes.
  7. Add 250-500 µL of LB or SOC medium to the cells.
  8. Incubate the cells at 37°C for an hour with shaking (~200 rpm) to allow the bacteria to recover and express the antibiotic resistance gene.
  9. After the recovery period, plate 50-200 µL of the transformation mix onto LB agar plates with the appropriate antibiotic.
  10. Spread the cells with an L-loop.
  11. Incubate the plates overnight at 37°C.

The day after, there should be colonies on the plates.

Figure 5. E. coli transformation plates.



To grow liquid cultures:

  1. Add 5 mL of LB to a culture tube.
  2. Add 5 µL of the appropriate antibiotic, based on the resistance gene present in the plasmid.
  3. Using a pipette tip, pick a single bacterial colony from a plate and inoculate the LB.

Incubate the culture under the appropriate conditions, shaking at 37ºC, during 24 hours.


To purify plasmid DNA using the MiniPrep method, you must start with a grown liquid culture.

  1. Pellet the bacterial cells by centrifuging the liquid culture.
  2. Resuspend the cell pellet in 250 µL of Buffer A1.
  3. Add 250 µL of Buffer A2, then gently mix by inverting the tube 6-8 times. Incubate at room temperature for no more than 4 minutes (do not vortex).
  4. Add 300 µL of Buffer A3 and gently invert the tube 6-8 times to mix (do not vortex).
  5. Centrifuge the mixture for 5-10 minutes at room temperature.
  6. Place a spin column in a 2 mL collection tube and load the supernatant from the previous step into the column.
  7. Centrifuge for 1 minute at 11,000 xg and discard the flow-through.
  8. Add 500 µL of Buffer AY, centrifuge for 1 minute, and discard the flow-through.
  9. Add 600 µL of Buffer A4, centrifuge for 1 minute, and discard the flow-through.
  10. Centrifuge again for 2 minutes to ensure all liquid is removed.
  11. Transfer the column to a new 1.5 mL Eppendorf tube, add 50 µL of Buffer AE, and incubate for 1 minute.
  12. Centrifuge for 1 minute to elute the plasmid DNA.

Note: The exact protocol may vary depending on the specific MiniPrep kit. This protocol is based on the NZYTech MiniPrep kit.

Figure 6. Centrifuge needed for the plasmid DNA purification.



First, you must grow a liquid culture of the desired C. acnes strain in liquid medium.

  1. Spin 50 mL of liquid culture at 4200 rpm for 10 minutes at 4ºC.
  2. Decant the supernatant carefully to remove it.
  3. Resuspend the cell pellet in 2 x 40 mL of ice-cold sucrose buffer.
  4. Centrifuge again at 4200 rpm for 10 minutes at 4ºC.
  5. Decant the supernatant.
  6. Resuspend the pellet in 1 mL of ice-cold sucrose buffer.
  7. Spin at 4200 rpm for 10 minutes at 4ºC.
  8. Decant the supernatant.
  9. Resuspend the pellet in 1 mL of ice-cold sucrose buffer and centrifuge at 10,000 rpm for 1 minute at 4ºC. Repeat this resuspension and centrifugation step three more times.
  10. After the final spin, resuspend the pellet in 100 µL of ice-cold sucrose buffer per 50 µL of competent cell suspension.

If you need to store the competent cells for future use you can split the competent cells made into smaller volumes. Freeze these immediately using liquid nitrogen and then store them at -80ºC.


Transforming C. acnes requires two days and the use of pre-prepared competent cells. Always work in a laminar flow hood to prevent contamination.

  1. In an Eppendorf tube, add the following in order:
    • 30 µL of sucrose buffer
    • 500-1000 ng of plasmid DNA
    • 10 µL of C. acnes competent cells
  2. Transfer the contents to a 1 mm cuvette and electroporate (1.5 kV, 25 µF, 400 ohm).
  3. Quickly add 100 µL of warm BHI medium and transfer the mixture back to an Eppendorf tube.
  4. Plate the mixture onto non-selective Brucella plates.
  5. Incubate the plates anaerobically at 37ºC for 24 hours.

The next day:

  1. Prepare Eppendorf tubes with 1 mL of BHI.
  2. Using a sterile swab, collect the entire contents from the plate and transfer them into the BHI.
  3. Centrifuge at 4,200 rpm for 5 minutes and discard the supernatant.
  4. Resuspend the pellet in 200 µL of BHI.
  5. Plate 100 µL on a Brucella + Erythromycin plate.
  6. Incubate the plates anaerobically at 37ºC for 6 days.
Figure 7. Our wet lab team doing the C. acnes transformation.



To grow liquid cultures for C. acnes:

  1. Resuspend the grown C. acnes plate into 10 ml of Brain Heart Infusion medium.
  2. Do a 1/10 dilution of the resuspension in more BHI medium, and then measure the OD 600. Values will go from 0.1 to 0.2, which means the original resuspension is around 1 to 2.
  3. The liquid culture will need to be at 0.1 OD 600 for correct growth. In a culture flask, add the right amount of the original resuspension and fresh BHI to achieve the desired volume at 0.1 OD 600.

The culture flask is going to be grown under anaerobic conditions, at 37ºC and shaking.


In an Eppendorf tube add:

  • 800 µL of grown culture.
  • 400 µL of glycerol at 50%

Freeze at -80ºC until needed for use.

Figure 8. Freezer where we kept our glycerol stocks.




Preparation of cell cultures

To perform a Western blot, you must first grow 30 mL of liquid culture of the cells you want to analyze. In our case, we worked with two different bacteria: E. coli and C. acnes.

  • E. coli liquid cultures need to be grown for 24 hours.
  • C. acnes liquid cultures must be grown for 3 days.

You also need to determine whether you're interested in analyzing protein expression in the bacterial pellet or the supernatant:

  • For E. coli, we focused only on the pellet.
  • For C. acnes, both the supernatant and pellet were of interest.

Protein Precipitation

Supernatant:

  1. Add 100 µL of 100% TCA (Trichloroacetic acid) per mL of supernatant.
  2. Precipitate the protein on ice for 2 hours.
  3. Centrifuge at 10,000 xg for 20 minutes at 4°C.
  4. Carefully aspirate the supernatant.
  5. Wash the pellet with ice-cold 100% acetone, then centrifuge at 10,000 xg for 5 minutes at 4°C.
  6. Repeat the washing step twice.
  7. Air-dry the pellet and resuspend it in 100 µL of PBS.

Pellet:

  1. Add 2 mL of PBS, resuspend the pellet, and centrifuge at 10,000 xg for 5 minutes at 4°C. Repeat this 2 more times.
  2. Resuspend the pellet in 1 mL of PBS.
  3. Transfer the resuspended pellet to a Precellys tube (for mechanical lysis using a homogenizer).
  4. Centrifuge the Precellys tubes at 10,000 xg for 10 minutes at 4°C.
  5. Wash the pellet with ice-cold 100% acetone, then centrifuge at 10,000 xg for 5 minutes at 4°C.
  6. Repeat the washing step twice.
  7. Transfer as much supernatant as possible to a clean Eppendorf tube.
Western Blot centrifuge used.
Figure 9. Homogenizer for mechanical lysis being set up.


Western Blot Procedure

Protein quantification:

  • The BCA Protein Assay is used to quantify the total protein concentration in the sample. The kit consists of Reagent A and Reagent B, which are mixed in a 50:1 ratio. This is our working solution.
  • A standard curve of known protein concentrations is needed to calculate the concentration (ng) of our samples. BSA protein is prepared from 10 ng/uL to 0 ng/uL by dividing the concentration in half each time.
  • Measure the absorbance using a multiwell plate reader with a measuring wavelength of 562 nm, loading 10 µL of each sample along with 190 µL of working solution.
  • Use the standard curve equation to determine the total protein concentration of our samples.

SDS-Page

Sample preparation:

  1. Prepare the sample mix: 15 µL of sample + 5 µL of sample buffer (Final volume = 20 µL per well). It is important to add a positive control to our gel.
  2. Sample buffer preparation: LDS sample buffer (4x, 190 µL) + β-mercaptoethanol (10 µL).
  3. Heat the samples at 95°C for 5 minutes using a Thermoblock to denature the proteins.

Running buffer:

  1. Prepare the running buffer: 15 mL of MES buffer + 285 mL of distilled water.
  2. Pour the running buffer into the Western blot chamber.

Loading the gel:

  1. Load 10 µL of protein ladder (SeeBlue) into the first well and 20 µL of each sample into subsequent wells.
  2. Run the gel at 120 V for 1 hour and 20 minutes.
Loading the gel.
Figure 10. Loading the gel.


Wet Transfer

Transfer Buffer Preparation:

Mix the following:

  • 50 mL of 20X transfer buffer.
  • 850 mL of deionized water.
  • 100 mL of methanol (MeOH).

Prepare for the wet transfer:

  1. Cut 4 filter papers and 1 PVDF membrane to the size of the gel.
  2. Soak the sponges in the transfer buffer.
  3. Activate the membrane by soaking it in methanol for 30 seconds, rinse with water, and then place it in transfer buffer.
  4. Assemble the "sandwich" in the MiniBlot module, from negative to positive: sponge, filter paper, gel, membrane, filter paper, sponge.
  5. Place the "sandwich" into the MiniBlot transfer tank, add transfer buffer, and run at 20 V for 1 hour.

Extract the membrane, never touching it with our hands, and place it in a small container, in this container:

  1. Prepare the blocking solution by mixing 2 g of milk powder with 40 mL of TBS-T.
  2. Block the membrane with 4% non-fat milk in TBS-T (TBS + 0.1% Tween 20) for 1 hour.
  3. Add the primary antibody (mouse anti-histidine tag) at a 1:800 dilution in 4% non-fat milk.
  4. Incubate overnight at 4°C.
  5. Wash the membrane 3 times with TBS-T for 10 minutes each.
  6. Add the secondary antibody at a 1:1000 dilution in 4% non-fat milk and incubate for 1 hour at room temperature.
  7. Wash the membrane 3 times with TBS-T for 10 minutes each.

Chemiluminescence Detection:

  1. Mix the luminol/enhancer solution and peroxide solution in a 1:1 ratio.
  2. Apply the solution above to the membrane and use a Biorad Imaging System to visualize the WB membrane with the corresponding bands.