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LB Medium Preparation - 1000 mL

  • Yeast Extract-5g
  • Tryptone-10g
  • Sodium chloride-10g
  • Agar-150g (if solid)
  • Water

Figure 1: PCR procedure.

Figure 2: PCR setup.

Restriction Enzyme Cutting

Figure 3: Restriction enzyme cutting procedure.

Patch and Small-Scale Culture

  1. Pick a colony by toothpick then “patch” on another agar plate as bacteria stock. Incubate in 37ºC for ≈14-16 hours.
  2. Dip the toothpick into 5 mL LB (with antibiotic) as small scale culture for plasmid extraction. Incubate in 37ºC at 170 rpm for ≈14-16 hours.

Gel Preparation - 50 mL

  • TAE buffer 50ml
  • 5 g agrose
  • Heat for 1 minute
  • 5 μL goldenview color dye

Transformation

  1. Take competent cell (E. coli) out of refrigerator, melt on ice.
  2. Add 10 μL ligation product to the melted competent cell, mix slightly, rest on ice for 30 min.
  3. 42℃ heat shock for 90s and place back on ice for 2-3 mins.
  4. Add 600 μL LB medium, and place in shaker for 45 mins (37℃, 220rpm)
  5. Centrifuge activated bacterial cells for 2 mins (6000 rpm)
  6. Discard 600 uL of waste solution.
  7. Transfer the remain 100 uL solution and apply on petri dish with LB.
  8. Place petri dish in incubator (37℃) overnight for culture.

Plasmid Extraction

  1. Place a CP3 rotary column in a clean collection tube and add 500μl of BL buffer. Centrifuge for 1 minute (12,000 rpm). Discard the waste in collection tube and return the rotary column back in the collection tube.
  2. Add 1-5 ml of bacterial cells cultured overnight in a microcentrifuge tube, and centrifuge for 1 minute (12,000 rpm). Discard the waste fluid.
  3. Add 250μl of P1 buffer and resuspend the bacteria.
  4. Add 250 μl of P2 buffer and mix gently with 6-8 inverts.
  5. Add 350μl of P3 buffer and mix gently 6-8 times inverts. The solution should be cloudy. Centrifuge for 10 minutes (12000rpm).
  6. Transfer the supernatant to CP3 spin column and place in the collection tube. Centrifuge for 30 – 60 seconds (12,000 rpm). Discard waste in collection tube, place CP3 spin column back in collection tube.
  7. Add 600μl of PW buffer in CP3 spin column and centrifuge for 30 – 60s (12,000 rpm). Discard waste in collection tube and return CP3 spin column back in collection tube.
  8. Repeat step 7.
  9. Place CP3 spin column in collection tube and centrifuge for 2mins (12,000 rpm) to clean remaining PW buffer. Open the CP3 spin column lid and rest in room temperature to evaporate remaining ethanol.
  10. Place the CP3 spin column in a clean microcentrifuge tube. Add 50-100 μl of EB buffer to the center of the CP3 spin column, and rest for 2 minutes, then centrifuge for 2mins (12,000 rpm).

SDS-PAGE

Reagents

  • 30% acrylamide solution: 29 g acrylamide, 1 g methylene acrylamide, dissolved to a volume of 100 mL at 37ºC, and stored in a brown bottle at 4ºC
  • Separation gel buffer (1.5 mol / L Tris-HCl): 18.2 g Tris, concentrated hydrochloric acid to pH 8.8, distilled water to a volume of 100mL
  • Concentrated gel buffer (0.5 mol / L Tris-HCl):
    • 6.0 g Tris, concentrated hydrochloric acid to pH 6.8., distilled water to a volume of 100mL
    • 10 × SDS-PAGE running buffer:
    • 30.2g Tris base, 188g Gly, 10g SDS to a volume of 1L;

Gel Preparation

  1. Clean and completely dry the plates, combs, and any other experiment materials. Assemble the system according to manufacturer’s instruction.
  2. Prepare the separating gel solution according to the following formula. Ammonium persulfate and TEMED are added when the gel is ready to be polymerized, stir gently, then immediately apply to the sandwich until the gel height is lower 1.5 cm than the short plate. Allowing the gel to polymerize 30~60min by covering gently with a layer of water (1cm).
  3. Until a sharp optical discontinuity at the water/gel interface is visible, pour off the water.
  4. Prepare the stacking gel solution in a similar way to the separating gel. Insert the comb and allow the gel to polymerize completely.

Figure 4: Gel reagents.

ComD ComE and CotA expression test

  1. Pick a colony by toothpick then dip the toothpick into 5 ml LB (with antibiotic) as small scale culture for protein expression test. Incubate in 37ºC at 220 rpm for ≈4-6 hours until OD600 = 0.6
  2. Induce the sample bacteria with 2.5μl IPTG(1M) in 5ml cultured medium. Incubate the medium in 22ºC at 170 rpm overnight.
  3. Centrifugal bacterial collection of cells. Centrifuge at 4ºC and 12,000 rpm for 1 minute.
  4. Add PBS to resuspend the cells.
  5. Take 30 μL suspension and add 10 μL 4 X protein sample buffer.
  6. Heat at 100ºC for 5 minutes.
  7. Electrophoresed on SDS-PAGE.180 V for 20 minutes, then 220 V until the band goes to the bottom.

Coomassie Blue Staining

  1. Add appropriate commassie solution to the SDS-PAGE
  2. Shake at the room temperature for 30 minutes
  3. Remove the dye, then wash the gel with Bleaching liquid

CotA dye-degradation test

The test dye of the experiment is reactive red
  1. Activate glycerin bacteria: take 1 mL of LB liquid medium without antibiotics, add 20 μL of glycerin bacteria (which has been transferred into plasmid containing CotA or ComD and ComE-CotA), and culture at 37ºC and 220 rpm for 5 hours.
  2. Take 500 μL of activated bacterial fluid in 40 mL different resistant LB medium (dependents on the group) overnight until OD600 = 0.6
  3. The bacterial solution was centrifuged at 4000 rpm 4ºC for 10 min, then resuspended with 5ml kanamycin resistant LB liquid medium, and centrifuged at 4000rpm 4ºC for 10 min, repeated another time; 30 mL of LB liquid medium containing kanamycin resistant was resuspended.
  4. 30 μL Triton X-100 (the final concentration of Triton X-100 is 0.1%) was added to 30 mL LB liquid medium containing kanamycin.
  5. Shaking at E. coli cells at 30ºC and 220 rpm for 30 min to increase their permeability.
  6. Centrifugation at 4000rpm 4ºC for 10 min, suspension with 5 mL LB liquid medium, centrifugation at 4000rpm 4ºC for 10 min then repeated another time.
  7. 25 mL LB liquid medium was used to resuspend the bacteria.
  8. Add the induction (iptg or csp) and RR color
  9. Each group was divided into 1 mL centrifuge tubes and 2 mL centrifuge tubes. The supernatant was centrifuged at 37ºC, 150 rpm for 0h, 4h, 8h, 12h, 24h, 36h, 48h, 12000 rpm for 1 min and then centrifuged to determine its absorbance at a wavelength of 530nm.

References

[1] https://www.neb.com/products/c2530-bl21-competent-e-coli

[2] https://www.goldbio.com/product/14438/dh5-alpha-chemically-competent-e-coli-cells