Preparation of Competent E. coli

1. Inoculate 2 mL of LB with a single E. coli colony. Incubate the preculture overnight at 37°C and 250 RPM.
2. In the morning, inoculate 500 mL of LB with 1 mL of saturated preculture. Incubate at 37°C and 250 RPM until OD₆₀₀ = 0.5.
3. Every step from now on should be completed on ice in a 4°C cold room. Transfer the culture to two prechilled sterile 250 mL centrifuge tubes.
4. Centrifuge at 2700g and 4°C for 10 minutes. Discard the supernatant and place the pellets on ice.
5. Resuspend each pellet in 10 mL cold 0.1 M CaCl₂. Pool into one prechilled sterile 50 mL centrifuge tube.
6. Centrifuge the cells at 2000g and 4°C for 5 minutes. Discard the supernatant and resuspend in 10 mL CaCl₂. Set on ice for 30 minutes.
7. Centrifuge the cells at 2000g and 4°C for 5 minutes. Discard the supernatant and resuspend in 2 mL CaCl₂. Leave at 4°C overnight.
8. The following day, add 70 µL of dimethyl sulfoxide (DMSO) and gently mix. Using liquid nitrogen, snap-freeze 50 µL aliquots of competent cells. Store at -80°C.

Polymerase Chain Reaction (PCR)

1. Combine 300 ng DNA, 25 µL 2x PCR Master Mix, 1 µL of 10 µM forward primer, 1 µL of 10 µM reverse primer and PCR-grade water up to 50 µL in thermal cycler snap tubes (ThermoFisher, AB2005).
   1. Add water first, then master mix, then primers, and lastly DNA.
   2. Make 100 µM stocks of primers in 10 mM Tris-Cl (pH 8) when they arrive, and then dilute tenfold with water for 10 µM working stocks.
   3. We used Quick-Load Taq 2x Master Mix, which contains Taq DNA Polymerase, its buffer, and dNTPs.
2. Place reaction mixture in thermal cycler under the following conditions:
   1. Initial Denaturation: 95°C for 30 seconds.
   2. 35 cycles of
      1. Denaturation: 95°C for 30 seconds.
      2. Annealing: 62°C for 60 seconds (varies with primers).
      3. Extension: 68°C for 120 seconds.
   3. Final extension: 68°C for 5 minutes.
   4. Hold: 4°C indefinitely.
3. All thermal cycler conditions change with the DNA polymerase, primers, and template. After testing our primers with a low-fidelity enzyme master mix, we used a high-fidelity enzyme master mix to amplify our sequences. The high-fidelity enzyme had higher accuracy but lower processivity, so it needed longer extension times and higher temperatures.
   1. Use this site to calculate optimal annealing temperatures (https://tmcalculator.neb.com/#!/main).
4. PCR products were purified from reaction mixtures with PCR Cleanup Kit (Qiagen, 28104).

Restriction Digeset

1. Add 300 ng DNA, 3 µL 10x Enzyme Buffer, 1 µL of each restriction enzyme (20,000 U/mL), and nuclease free water up to 30 µL total reaction volume.
   1. Use the NEBuffer Activity/Performance Chart with Restriction Enzymes to find the optimal buffer for the enzyme(s) being used, i.e. r2.1 for NdeI.
   2. Calculate volumes to add beforehand; add water first, then buffer, DNA, and lastly enzymes.
2. Let the reaction mixture incubate at 37°C for one hour, then store at -20°C until use.
   1. Ideally, digestion products should be used immediately to avoid star activity.

DNA Gel Electrophoresis

1. Prepare gel tray by adding end gates via lab tape, using your nails to create a firm seal.
2. Create a 1% w/v agarose gel solution by adding 0.5 g agarose to 50 mL 1x TAE solution. Place in a lab microwave until the solution begins to boil (about 50 seconds). When the solution has cooled to room temperature, add 5 µL 10,000x GelRed (Biotium, 41003) and swirl to mix.
   1. For smaller DNA fragments (less than 1 kb), we used a 1.3% agarose gel for clearer band separation.
3. Pour agarose gel solution into gel tray. Add comb to create wells. Let cool until solidified (about 30 minutes).
   1. Check after 10 minutes that end gate tape seal is holding.
4. Remove tape seal from gel tray and place gel tray in the buffer chamber. Pour into buffer chamber enough running buffer solution (TAE) until top of gel is covered.
5. Prepare samples to load by spotting 1 µL 6x Purple Gel Loading Dye (New England Biolabs, B7024S) onto parafilm, pipetting 12 µL sample onto the spot, mixing, and pipetting 12 µL sample into a well.
   1. We initially followed manufacturer recommendation of 1 µL dye per 5 µL sample but found that our sample bands were still clear with less dye!
   2. We used 1Kb Plus DNA Ladder (Invitrogen, 10787018) diluted 1:4 with water as a DNA ladder in all our gels.
6. Run the gel by covering buffer chamber with lid such that the anode (red) (positive charge) is oriented at the bottom of the gel with the wells at the top. Turn on the power supply and set to constant voltage at 60 mV.
   1. A good indication that voltage is applied to the gel is a stream of bubbles appearing in the running buffer solution at the cathode side (black) (negative charge).
   2. Check back after 5 minutes to ensure that dye bands have moved from wells towards bottom of gel.
7. When the dye bands reach ¾ of the way towards the bottom, turn off power supply and remove gel from buffer chamber. Visualize DNA bands on a UV table with low intensity (high UV provides a clearer image but induces mutations faster), or image DNA bands on a gel imager, such as a Gel Doc EZ Imager (Bio-Rad).

Ligation

1. Perform restriction digest on insert and vector as described above.
2. Use a DNA Cleanup Kit (Monarch, T1030S) to purify DNA from reaction.
3. Characterize concentration of digested fragments with NanoDrop (Thermo Scientific, 13-400-519).
4. Combine digested insert, digested vector, 2 µL 2x ligation buffer (New England Biolabs, B0202S), 1 µL DNA ligase (400,000 U/mL) (New England Biolabs, M0202S), and water up to 20 µL final volume in a 50 µL Eppendorf tube.
• Add pre-calculated volume of water first, then buffer, then DNA, then enzyme.
• Insert and vector should have been digested with the same pair of enzymes.
• Insert should be added in a 3:1 molar ratio to vector.
5. For example, if there is a 10 ng/µL digested insert solution and 30 ng/µL digested vector solution, and the insert and vector are 1 kb and 3 kb respectively, then 3 µL insert for every 1 µL vector.
6. Let reaction mixture incubate for 3 hours at 16°C in thermal cycler set to continuous temperature.
7. Directly transform competent cells with 5 ng DNA from ligation mixture.
• Competent cells with lower transformation efficiency will require more DNA to observe transformant colonies.

Transformation of Chemically Competent Cells

1. Thaw competent cells on ice. When barely thawed, split cells into 25-50 µL volumes in 1.5 mL sterile tubes. Add 1 pg to 100 ng (< 5 µL) of plasmid DNA to the cells and mix by gently flicking 4-5 times.
2. Place the cells on ice for 30 minutes. Do not mix.
3. Heat shock the cells at 42°C for 45 seconds. Immediately place on ice for 2 minutes.
4. Add room-temperature SOC outgrowth medium (New England Biolabs, B9020S) to 1 mL total volume per tube. Incubate at 37°C and 250 RPM for 1 hour.
5. While incubating, warm antibiotic selection plates to 37°C.
6. Depending on the transformation efficiency of the cells, it may be necessary to dilute the cells in SOC. Using sterile glass beads, spread 50-200 µL of cells onto the warmed plates.
7. Incubate at 37°C for 16-20 hours.
8. Transformation efficiency is calculated as the number of distinct colonies plated per µg of DNA.

Preparing Glycerol Stocks of Strains

1. Select colony from plate with pre-sterilized inoculator loop or flame-sterilized wooden dowel, and dissolve in 2 mL Luria-Bertani Medium (LB) to create an overnight culture.
   1. Add antibiotic if cell line contains a plasmid to prevent plasmid loss.
2. Let inoculated culture incubate in 37°C incubator shaking at 250 RPM for 12 hours or overnight.
3. Into a cryotube, pipette 1 mL overnight culture and 1 mL 50% glycerol. Label cryotube and place in –80°C freezer for storage.
   1. Final glycerol concentration can range from 15-50%. 25% is ideal for scraping for future use.

Protein Expression

1. Start overnight culture of strain containing plasmid to be expressed in LB medium at 37°C.
2. In the morning (after ~12 hours), dilute overnight culture 1:10 by adding 400 µL overnight culture to 4 mL LB broth, as well as 1x final concentration antibiotic to which plasmid contains resistance.
3. Periodically check OD₆₀₀ using spectrophotometer blanked with LB medium from the same bottle used to prepare the culture. When OD₆₀₀ = 0.6, divide culture in half into separate culture tubes (2 mL each). Induce protein expression in one tube by adding 1 µL 0.8 M isopropyl thiogalactopyranoside (IPTG) for final concentration 0.4 mM IPTG.
   1. Our two plasmids use a T7-inducible promoter system, so we induce with IPTG. Different inducible promoter systems may use different molecules.
   2. The non-induced tube serves as a negative control.
4. After four hours, pellet cells by centrifuging at 13,000g for 1 minute and remove supernatant. Use in downstream applications.
   1. An inducer concentration, temperature, and induction period scan can be used to determine optimal protein expression. In separate experiments, vary the amount of inducer used, the temperature at which protein is expressed, and the amount of time for which protein is expressed. For example, we adapted our protocol from a different expression system and found that we could produce more protein by inducing with less IPTG at a lower temperature for longer periods of time. You can also try varying the OD₆₀₀ at which you induce expression.

Cell Fractionation

1. Dilute overnight culture 1:10 into 14 mL culture. When OD₆₀₀ = 0.6, divide culture into 2 mL (negative control) and 12 mL portions. Induce protein expression at OD₆₀₀ = 0.6.
2. Take 1 mL of cells from induced and non-induced cultures. Pellet each separately at 13,000g for 1 minute at room temperature. Remove supernatant and resuspend pellets in 30 µL 1% w/v SDS and 102 µL 2x Sample Buffer. Heat for 3 minutes in boiling water. Label tubes WC-I and WC+I, respectively, and store at –20°C.
   1. Use a vacuum or micropipette to aspirate off all supernatant.
3. Pellet 9 mL induced culture, remove supernatant, and resuspend in 1 mL Sonication Buffer.
4. Sonicate 3 x 15 seconds on ice with 20 second pause between sonication cycles. Power at 24 W. Microprobe used.
   1. Save 5 µL pre-sonicate to compare against sonicate with phase-contrast microscopy. Use this technique to guarantee cell lysis before continuing.
   2. Inclusion bodies will appear as clear circles that refract light. If your protein is present in inclusion bodies, you will need to modify the purification protocol from what is given here.
5. Create protein sample from sonicate by adding 75 µL sonicate to 25 µL 1% w/v SDS and 250 µL 2x Sample Buffer. Heat for 3 minutes in boiling water and label tube Son. When loading onto SDS-PAGE gel, use 2.38x the volume of this sample relative to volumes of samples prepared in other steps. Store at –20°C.
6. Centrifuge remaining sonicate at 4°C for 10 minutes at 13,000g. Remove 875 µL supernatant and keep on wet ice. Aspirate or pipet away remaining supernatant.
7. Create protein sample of supernatant by adding 25 µL supernatant to 25 µL 2x sample buffer and heating for 3 minutes in boiling water.
8. Create protein sample of pellet by resuspending it in 875 µL 0.3% w/v SDS, adding 875 µL 2x sample buffer, and boiling in water for 3 minutes.
9. Spin down the 875 µL saved supernatant in ultracentrifuge at 100,000g at 4°C for 15 minutes.
   1. Pre-chill the rotor. Use this site (https://www.sciencegateway.org/tools/rotor.htm) to calculate the correct RPM for your chosen rotor. For example, 100,000g in a TLA-120.1 Beckman rotor corresponds to 53,000 RPM.
   2. Don’t forget to use ultracentrifuge tubes that won’t shatter at high g-force.
10. Collect high speed supernatant and save for downstream application.
11. Create protein sample of high-speed supernatant by adding 25 µL high speed supernatant to 25 µL 2x sample buffer and boiling in water for 3 minutes.
12. Create protein sample of high-speed pellet by resuspending in 850 µL 2x sample buffer and adding 425 µL 0.3% w/v SDS. Boil in water for 3 minutes.
13. Run an SDS-PAGE gel of each protein sample made in this protocol. Monitor the presence of your expressed protein through each step and verify it is the portion that you use in downstream applications.

SDS-PAGE

1. Electrode buffer should completely cover the openings at the top and the bottom of the plates, in order to properly allow current to flow through the gel.
2. Load up to 20 µL of protein sample per well. For the colorimetric ladder, load less than 5 µL.
   1. For the ladder, we used Precision Plus Protein Dual Color Standards (Bio-Rad, 161-0374).
3. Start electrophoresis at 5 mA per gel.
4. When the protein has passed through the stacking gel, turn the current up to 15-18 mA per gel.
5. Wait until the dye front has passed all the way through the gel to allow for maximum separation of protein.

1. After opening the plates, carefully cut off the stacking gel with a razor and dispose of it. Use round-tipped tweezers to gently place the running gel in a small box or tray. To save staining and destaining solution, the bottom of the box should not be much larger than the gel.
2. Add enough Fairbanks I to cover the gel. Place on rocker overnight.
3. In the morning, replace Fairbanks I with Fairbanks II. Place on rocker for 6 hours.
4. Replace Fairbanks II with the destaining solution. Place on rocker for an hour.
5. Replace destaining solution with fresh destaining solution. Place on rocker for another hour.
6. Image.

Western Blot

1. Assemble the cassette in the following order after soaking each piece in transfer buffer: sponge, filter paper x2, nitrocellulose membrane, SDS-PAGE gel, filter paper x2, sponge.
   1. The membrane should only be touched with tweezers to prevent unintentional transfer of protein from external sources.
2. Fill the electrophoresis box with transfer buffer. When placing the cassette in the box, the nitrocellulose should be on the cathode side (the protein is negatively charged).
3. In a cold room (4°C) with a stir bar in the electrophoresis box, run at 60 V for 4 hours.
4. Carefully remove the nitrocellulose membrane and place into a plastic box/tray. Add Ponceau S stain and place on rocker for 5 minutes. This will quickly show any protein on the membrane.
5. Remove the Ponceau S and use a squirt bottle with purified water to get off any excess stain.
6. Using a soft pencil, mark ladder and lanes. Cut away unnecessary membrane with a razor blade. Place the membrane into the smallest container it will fit into, to conserve reagents.

7. Wash for 5 minutes with Tris buffered saline (TBS). Wash with 5% w/v instant milk (Carnation brand) for 30 minutes to block the membrane. Wash twice with Tris buffered saline with Tween 20 (TBST) for 5 minutes each.
8. Add primary antibody to 5 mL of TBST. Wash for 2 to 3 hours minimum (overnight is fine).
   1. Use manufacturer recommendations for antibody dilution.
9. Wash with TBS, then TBST twice, then TBS for 5 minutes each.
10. For every wash step from now on, cover in tinfoil to prevent photobleaching of the fluorophore conjugated to the secondary antibody.
11. Add secondary antibody to 5 mL of TBST. Wash for 30 minutes – more than 30 minutes risks having too bright a background.
    1. Use manufacturer recommendations for antibody dilution.
12. Wash with TBS, then TBST twice, then TBS for 5 minutes each.
13. Wash with TBS twice for 10 minutes each.
14. Image on Gel Doc EZ Imager with protein gel and correct stain selected.

Affinity Purification

(Adapted from Roche Ni NTA protocol https://www.studocu.com/row/document/)

1. In 4°C cold room, add 1 mL Ni-NTA resin slurry to the column. Equilibrate the column by adding 10 mL equilibration buffer and pipetting to mix with the beads. Wait 15 minutes for beads to settle back down to the bottom of the column.
2. Under gravity flow, apply sample to the column when equilibration buffer nearly reaches the resin bed. Make sure that the resin bed never runs dry.
1. Save a small portion of the sample as a control.
2. If sample has not been ultracentrifuged, it might clog the column.
3. After the sample has entered the resin, add 10 mL wash buffer. Begin collecting 2 mL fractions of effluent, starting with the flow through (obtained after adding sample to column but before adding wash buffer).
4. Elute nonspecifically bound proteins with 10 mL elution buffer 1.
5. Elute specifically bound protein with 10 mL elution buffer 2.
6. Elute all bound proteins with 10 mL elution buffer 3.
1. The elution buffer series contains an increasing concentration of imidazole, a molecule which replaces the His₆ tag that binds the Ni²⁺ ions.
7. Run a SDS-PAGE gel of all fractions to identify fractions containing pure protein of interest.
1. You can optimize elution conditions for your specific protein by changing the concentration of imidazole in each elution buffer. For example, increase the imidazole concentration in elution buffer 2 if you notice there is protein of interest in elution buffer 3, or decrease the imidazole concentration in elution buffer 1 if you notice protein of interest is eluting too early in elution buffer 1.
8. Once protein of interest is confirmed in elution fraction, remove imidazole by dialysis.
1. Re-hydrate MWCO 1000 dialysis tubing (Spectrum Laboratories, Inc., 086802C) in dH₂O for 15 minutes.
2. Add fraction containing protein of interest to tubing and remove air bubbles before clamping off tubing.
3. Place in 2 L of 5 mM Sodium Phosphate Buffer (pH 7), made by diluting 33.3 mL 0.6 M Sodium Phosphate Buffer (pH 7) in up to 2 L of dH₂>O.
4. After 4 hours, replace solution with another 2 L 5 mM Sodium Phosphate Buffer pH 7.
5. After another 4 hours or overnight, remove sample from solution.
9. Snap freeze by adding 5% glycerol v/v and placing in liquid nitrogen. Store in –80°C.

Disk Diffusion Assay

1. Prepare M16 + 5% glucose (pH 6) plates.
   • Include recipe for these plates.
   • Store plates at 4°C.
2. Prepare antibiotic disks by adding 10 µL solution to sterile disks (Becton, Dickinson and Co., 231039) and letting the disks dry in a 37°C incubator for 4 hours.
   • Use solvent as negative control.
   • Use 5 mg/mL kanamycin as positive control.
   • 5 mg nisin (Sigma Aldrich, N5704) was dissolved in 1 mL 50 mM Sodium Phosphate Buffer to prepare 5 mg/mL stocks.
3. Let plates dry for one hour in a 37°C incubator by placing them upside down with the agar plate itself placed at an angle in the dish, such that the lid is cracked open.
4. Prepare 1 mL of cell culture by dissolving scraped M. luteus colonies from a LB plate in 0.9% NaCl solution.
   • Measure the absorbance with a spectrophotometer and dilute the sample until OD₆₀₀ = 0.2.
5. Pipette 800 µL of cell culture onto dried plates, use a sterilized spreader to distribute the liquid equally across the plate, and let the plate incubate right side up in a 37°C incubator for 2 hours or until the liquid has been absorbed.
6. Sterilize metal forceps with ethanol and flame before picking up antibiotic disks and placing them onto the plates. Let the plates incubate in a 37°C incubator for 24-48 hours, or until zones of inhibition can be measured.
7. Measure zones of inhibition with a ruler by taking three diameter measurements for each zone and averaging them.
   • If no zone of inhibition forms, record value as 0 cm and not the diameter of the disk itself.

Franz Diffusion Assay

1. Allow porcine skin (Stellen Medical L LLC, Fisher Scientific, NC1275387) to thaw at room temperature for 30 minutes.
2. Cut a 2.5 x 2.5 cm² skin sample using a sterile scalpel.
3. Measure skin resistivity using a multimeter
1. Place one electrical probe on the stratum corneum side of the skin.
2. Place the other electrical probe on the dermal side of the skin.
3. Resistivity values less than 187.5 kΩ (30 kΩ/cm²) were considered unfit for further experimentation.
4. Place a stir bar in the receptor compartment of the Franz Diffusion cell (Figure 2).
5. Stretch the skin over top of the receptor compartment ensuring there is little to no folding on the surface of the skin. Then, place the donor compartment over the skin and clamp together.
6. Load 9 mL of Phosphate-Buffered Saline (PBS) (pH 7.4) ensuring that no air bubbles form underneath the surface of the skin.
1. Tip: Tilt the clamped Franz Diffusion cell such that the sampling port is pointing upwards.
7. Place a dish with stir bar and water on a stir plate in an incubator.
8. Turn on stir plate and allow temperature of the water to increase to 37°C.
9. Place the Franz Diffusion cell, fitted with porcine skin and PBS, in the dish in the incubator.
10. Wait 30 minutes to allow temperatures to increase to 37°C.
11. Apply 1 mL of donor solution to the donor compartment.
12. Take 20 µL samples from sampling port at regular intervals for 24 hours.
13. Measure the absorbance of the sample at 274 nm.
14. Determine concentration of receptor compartment using known absorbance values.
15. Replace the extracted volume with PBS, once again ensuring that no air bubbles form underneath the surface of the skin.

Figure 3. Franz diffusion cell components. (1) Sampling port, where samples are extracted from the receptor compartment. (2) Porcine skin, serves as the membrane through which permeable substances move between the donor and receptor compartments. (3) Donor compartment, where the donor solution is applied. (4) Clamp, used to hold the skin in place between donor and receptor compartments. (5) Receptor compartment, where substances that permeate the membrane are collected. (6) Stir bar, used to ensure that the receptor compartment concentration is uniform.

Figure 4. Experimental setup of the Franz diffusion cell. Placed in a water bath on a stir plate inside an incubator. Cork used to keep the cell upright during the experiment.