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Protocol

Molecular cloning-related experiments

Gibson assembly


Protocol for 2-3 Fragment Assembly

  1. Prepare 10 μl mixture of linear vector: insert = 1:3 (pmole) on ice.
  2. Add 10 μl Gibson Assembly Master Mix (2X) to 10μl mixture on ice.
  3. Incubate the reaction at 50°C for 50 mins.
  4. Transform the E. coli with the Gibson assembly mixture.

Prepare LB agar plate


Protocol

  1. Mix 20 g/L LB Broth and 15 g/L agar in a serum bottle.
  2. Autoclave at 121°C, 2 atm for at least 20-minute liquid cycle.
  3. Heat the molten gel mix by partially submerging the bottle in the 60 ℃ water bath.
  4. Add antibiotics with proper concentration, and mix by swirling.
  5. Pour into plates (~15 ml/plate), covering the surface and avoiding bubbles.
  6. Let plates cool with the lids ajar.

Transformation


Materials

  • Competent cells ECOS™ FYE 207/FYE678(YB Biotech).

Protocol

  1. Add competent cells with 3 times the ligation reaction volume for the ligation product and incubate on ice for 5 mins. For intact plasmid, mix 50 ng plasmid with 10 μl competent cell.
  2. Heat-shock the competent cells at 42°C for 45 seconds.
  3. Plate to LB-agar plate.

Colony PCR


Protocol

-Preparation

  1. For each colony, prepare one tube for PCR and one for saving bacterial stock.
  2. Add 9 μl ddH2O into each 200 μl PCR tube.
  3. Add 80 μl TB with antibiotics into each 1.5 ml tube to save the bacterial stock.
    • Pick colonies for PCR and stock
      ▫ Using a tip, pick a single colony and gently mix it in 9 μl of ddH2O in the PCR tube, immediately followed by mixing it in the TB with antibiotics in the 1.5 ml tube.
    • Prepare PCR mix (total volume: 15 μl)
      ▫ For each colony, prepare a PCR mix with the following table.
component volume
GoTaq® Green Master Mix 10 μl
Primer (Forward) 0.5 μl
Primer (Reverse) 0.5 μl
ddH2O 9 μl
Total 20 μl

-Add 20 μl mixture to each PCR tube.

-Run PCR

Bacteria stock


Protocol

  1. Mix the 50% glycerol and bacterial culture with 1:1 volume.
  2. Store the mixture in the -80°C freezer as bacterial stock.

Agarose Gel extraction (Geneaid No.DFH300)


Protocol

Step 1: Gel Dissociation

  1. Transfer 300 mg (about 3 wells) of gel slice to 1.5 ml microcentrifuge tube.
  2. Add 500 μl of Gel/PCR Buffer, vortex, and incubate at 55-60°C for 10-15 minutes.
  3. Add 10 μl of 3M Sodium Acetate if turned to purple.

Step 2: DNA Binding

  1. Place the DFH column in 2 ml collection tube.
  2. Transfer 800 μl sample mixture to the DFH column.
  3. Centrifuge (14-16000) for 30 sec.
  4. Discard the flow-through and place the DFH column back in the 2 ml collection tube.
  5. Repeat the DNA Binding step if the sample mixture is more than 800 ul.

Step 3: Wash

  1. Add 400 μl W1 Buffer into the DFH column.
  2. Centrifuge (14-16,000 x g) 30 sec then discards flow-through.
  3. Add 600 μl Wash Buffer (make sure alcohol is added) into the DFH column, stand at room temperature for 1 min, centrifuge (14-16000 x g) for 30 sec then discard flow-through.
  4. Place the DFH column back and centrifuge (14-16000 x g) for 3 min to dry the column matrix.

Step 4: DNA Elution

  1. Transfer the dried DFH column to a new 1.5 ml microcentrifuge tube.
  2. Add 20 μl ddH2O into the center of the column matrix.
  3. Let stand for at least 2 minutes to ensure the water is completely absorbed.
  4. Centrifuge (14-16,000 x g) for 2 minutes to elute the purified DNA.

Plasmid extraction (Geneaid No.PDH300)


Protocol

  1. Draw 1.5 ml from a 2 ml bacterial culture tube into a new 1.5 ml tube, leaving the remaining 0.5 ml in the test tube.
  2. Centrifuge the new 1.5 ml tube for 1 minute at 14000 rpm. Use a pipette to aspirate the supernatant after centrifugation (be careful not to aspirate the plasmid layer at the bottom).
  3. Tilt the rack and use the tube to scrape it to disperse the bacteria in the tube.
  4. Add 200 μl of PD1 and vortex once to mix the liquid.
  5. Add 200 μl of PD2. Do not vortex to avoid DNA breakage. Instead, use two racks to sandwich the tube and shake it up and down 10 times.
  6. Insert the pipette tip beneath the liquid surface and add 200 μl of PD3.
  7. Centrifuge the tube for 3 minutes at 5000 RCF (relative centrifugal force) and place the PDH Column into a 2 ml Collection Tube. If proteins remain suspended in the upper-middle layer, centrifuge again for 3 minutes at 15000 rpm.
  8. Suck up 600 μl of the liquid containing DNA into the PDH column. Discard the 1.5 ml tube. (Do not aspirate the white proteins; if suspended proteins are aspirated, draw 400 μl, then centrifuge the tube for 3 minutes at 15000 rpm, and then aspirate the remaining 200 μl).
  9. Centrifuge the PDH column for 30 seconds at 15000 rpm and discard the flow-through.
  10. Add 400 μl of W1 to the PDH column, centrifuge for 30 seconds at 15000 rpm, and discard the flow-through.
  11. Add 600 μl of Wash Buffer to the PDH column, and centrifuge for 30 seconds at 15000 rpm (in the process, get a 1.5 ml tube ready). After centrifugation, discard the flow-through.
  12. Centrifuge the dry PDH column with no added liquid for 3 minutes at 15000 rpm. (ensure the PDH column is dry).
  13. Place the PDH column into a 1.5 ml tube, add 20 μl of Elution Buffer (can be replaced with 60°C ddH2O), and time for 2 minutes to ensure DNA binds to the Elution Buffer. (Elution Buffer can be replaced with 60°C ddH2O; it's recommended to heat ddH2O to 60°C for better DNA binding).
  14. Centrifuge the 1.5 ml tube containing the PDH column for 2 minutes at 13000 rpm.

Protein expression-related experiments

Bacterial protein induction


Preparation

  • Cultured 3 ml bacteria in TB-antibiotics at 37℃ overnight.

Refresh

  • Quantify and dilute the grown bacteria to an O.D. 600 value of around 0.05.
  • Refresh the diluted bacteria at 37℃ for 2 hrs.
  • Quantify the O.D. 600 value of refreshed bacteria. If the O.D. 600 value reaches 0.5~0.9, transfer 500 ul of refreshed bacteria to a 1.5 ml tube as the non-induction sample, If not, then put the refreshed bacteria back to 37℃ for one more hour.

Induction

  • Induced the protein expression in the rest of the refreshed bacteria by 0.2 mM IPTG at 37℃ for a few hours.

Storage

  • Non-induction and induction samples are centrifuged at 14000 rpm for 1 mins.
  • Remove the supernatant.
  • Directly store the pellet at -20℃, or lyse it with 100 μl lysis buffer and store it at -20℃.

Bacterial protein extraction


Solution preparation

component volume
50 mM Tris (pH 8.0) 2.5 ml
10% Glycerol 10 ml
0.1% Triton 0.05 ml
ddH2O 37.45 ml
Total 50 ml

Protocol

  1. Resuspend 1.5 mL pellet of bacteria cell culture in 0.1 ml lysis buffer.
  2. Incubate 30 minutes on the ice.
  3. Sonication 3x10" till the sample is no longer viscous.
  4. Centrifuge 14000 rpm for 10 min at 4°C.
  5. Collect the supernatant into a new tube.
  6. Store sups and the dried pellet at -20°C.

SDS-PAGE


Materials & protocol

Gel preparation

Lower Gel-For 2 Gel
Percentage 15% 12% 10% 7.5%
ddH2O 2.35 ml 3.35 ml 4.0 ml 4.85 ml
Tris buffer (1.5m, pH 8.8)+10% SDS 2.5 ml 2.5 ml 2.5 ml 2.5 ml
30% (29:1) Acrylamide: Bisacrylamide 5.0 ml 4.0 ml 3.33 ml 2.5 ml
10% APS 100 μl 100 μl 100 μl 100 μl
TEMED 10 μl 10 μl 10 μl 10 μl
Vtotal 10 μl

Upper Gel-For 2 Gel
Percentage 5% 4%
ddH2O 1.75 ml 1.85 ml
Tris buffer (1.5m, pH 8.8)+10% SDS 0.75 ml 0.75 ml
30% (29:1) Acrylamide: Bisacrylamide 0.5 ml 0.4 ml
10% APS 30 μl 30 μl
TEMED 5 μl 5 μl
Vtotal 3 μl

Solution Preparation

Fixing solution
Ethanol 150 ml
Glacial acetic acid 50 ml
ddH2O 300 ml
Total 500ml

Staining solution
Methanol 150 ml
Glacial acetic acid 50 ml
Coomassie brilliant blue 1 g
ddH2O 300 ml
Total 500 ml

Distaining solution
Methanol 150 ml
Glacial acetic acid 50 ml
ddH2O 300 ml
Total 500 ml

Gel preparation

  1. Wipe glass plates and spacers and assemble them in a gel casting apparatus.
  2. Mix components for the resolving gel and pour into the gel plate.
  3. Add 1000 μl isopropanol and ddH2O on the top of the resolving gel to flatten the resolving gel.
  4. Until the resolving gel is solidified, clean up the ddH2O on the resolving gel.
  5. Mix components for the stacking gel and pour into the gel plate.
  6. Insert the comb into the top of the spacers, and then wait for the gel to solidify.

Gel electrophoresis

  1. Add 1x running buffer to the running tank and put the cast gel into it.
  2. Load 10 μl marker into the first well and 20 μl protein samples per well.
  3. The gel will run at 80 V until the dye front is migrated into the running gel (about 20 minutes) and increase to 100 V until the dye front reaches the bottom (about 90 minutes).

Gel fixing, staining, and distaining

  • Put gel into gel-fixing solution for 1 hour.
  • Stain the gel with staining solution for 20 minutes.
  • Disdain the gel for about 1.5~2 hours until the bands are clear enough.

    • 5-HTP production related experiment

    1. Induced the protein expression in the rest of the refreshed bacteria by 0.2 mM IPTG at 37℃ for a few hours.
    2. Centrifuge samples at 1000 x g for 20 minutes. Collect the supernatant and assay immediately, or store samples in an aliquot at -20°C or -80°C for later use. Avoid repeated freeze-thaw cycles.
    3. After the kit is equilibrated at room temperature, add 50 μL of Standard Working Solution or 50 μL of sample to each well, immediately add 50 μL of 1x Biotinylated-Conjugate Working Solution to each well, mix well, incubate at 37°C for 60 minutes.
    4. Discard the liquid in the plate, add 200 μL 1x Wash Buffer to each well, and wash the plate 3 times. After patting it dry against clean absorbent paper, add 100 μL 1x Streptavidin-HRP Working Solution to each well and incubate at 37°C for 60 minutes.
    5. Discard the liquid in the plate, add 200 μL 1x Wash Buffer to each well, and wash the plate 5 times. After patting it dry against clean absorbent paper, add 90 μL TMB Substrate Solution to each well and incubate at 37°C for 20 minutes in the dark.
    6. Add 50 μL Stop Reagent to each well, and shake the plate on a plate shaker for 1 minute to mix. Record the OD immediately calculation of the results.
    concentration

    Carrier-related experiments

    E. coli growth curve


    Preparation

    1. Culture E. coli overnight (16 to 18 hours) at 37℃.
    2. Add 100 μL of the overnight E. coli culture to each of the three wells in a 96-well plate.
    3. Add 100 μL of TB+Amp to each of the three other wells in the same plate.
    4. Use Nanodrop to measure the OD600 value.
    5. Average the three OD600 values of the TB+Amp wells to obtain the background value.
    6. Average the three OD600 values of the overnight E. coli cultures and subtract the background value to obtain the initial E. coli OD600 value.

    Protocol

    1. Calculate the ratio of E. coli solution to TB+Amp based on the initial OD600 value, and dilute the E. coli solution until the OD600 value reaches 0.05.
    2. Incubate the diluted E. coli solution at 37°C and measure the OD600 value every 30 minutes for 12 hours.
    3. Plot the E. coli growth curve using the obtained data.

    Microencapsulation


    Preparation

    Refresh E. coli from the Stationary Phase into the Log Phase

    1. Refresh the E. coli culture from the stationary phase into the log phase.
    2. Collect 20 mL of E. coli in the log phase, with a concentration of 1 x 10⁷ CFU/mL.

    Preparation of Solutions

    1. Prepare a chitosan solution by dissolving chitosan in 1% acetic acid to achieve a final concentration of 1 mg/mL (pH 6.0).
    2. Prepare a sodium alginate solution by dissolving sodium alginate in 1M NaCl to achieve a final concentration of 2 mg/mL.
    3. Sterilize the chitosan and sodium alginate solutions at 121°C for 15 minutes.
    4. Prepare a 1% calcium chloride solution.

    Protocol

    Layer-by-Layer (LbL) Assembly Method

    1. Centrifuge 20 mL of log phase E. coli solution at 4000 rpm for 5 minutes.
    2. Remove the supernatant and add 5 mL of PBS.
    3. Centrifuge at 4000 rpm for 5 minutes.
    4. Repeat adding 5 mL of PBS to obtain E. coli pellets.
    5. Resuspend the pellet in 3 mL of PBS.
    6. Add 1.5 mL of chitosan solution to the 3 mL E. coli suspension, then place it in a 37°C incubator and shake for 10 minutes.
    7. Centrifuge at 4000 rpm for 5 minutes and remove the supernatant.
    8. Wash the pellet by adding 3 mL of PBS to remove unadhered chitosan.
    9. Centrifuge at 4000 rpm for 5 minutes and remove the supernatant.
    10. Resuspend the pellet in 4 mL of PBS.
    11. Add 0.5 mL of sodium alginate solution to the 4 mL E. coli suspension.
    12. Place in a 37°C incubator and shake for 10 minutes.
    13. Centrifuge at 4000 rpm for 5 minutes and remove the supernatant.
    14. Wash the pellet by adding 3 mL of PBS to remove unadhered sodium alginate.
    15. Centrifuge at 4000 rpm for 5 minutes and remove the supernatant.
    16. Repeat the steps of adding chitosan and sodium alginate one more time.
      Note: Add 0.5 mL of chitosan solution to the third layer's 4 mL E. coli suspension.

    Ionic Cross-Linking Method

    1. Add 4.8 mL of PBS to the centrifuged pellet to resuspend.
    2. Add 0.2 mL of 1% CaCl₂ solution to the 4.8 mL E. coli suspension.
    3. Place the suspension in a 37°C incubator and shake for 10 minutes.
    4. Centrifuge with 3 mL of PBS at 4000 rpm for 5 minutes to remove unadhered CaCl₂.
    5. Place the sample in a -80°C freezer for at least two hours to prepare for freeze-drying.
    6. Transfer the sample to a freeze dryer and process until the sample becomes powdery.

    Zeta Potential Detection


    Preparation

    1. Prepare samples of the following 5 stages, each suspended in 1 mL of PBS:
      • Stage 1: uncoated E. coli
      • Stage 2: E. coli-chitosan
      • Stage 3: E. coli-chitosan-alginate
      • Stage 4: E. coli-chitosan-alginate-chitosan
      • Stage 5: E. coli-chitosan-alginate-chitosan-alginate
    2. Measure the OD value of each suspended sample

    Protocol

    1. Open the application "Zetasizer".
    2. Add 1 mL of the sample into a cuvette.
    3. Place the cuvette into the Dynamic Light Scattering (DLS) machine.
    4. Set the sample parameters in the application.
      Meterial DH5α
      Dispersant 0.9% NaCl
      Temperature 25℃
      Cuvette DTS1070
      Measurement duration automatic
      Number of measurement 3
      Delay between measurements 30 seconds
    5. Start detecting the zeta potential for each sample.
    6. Import the data for each sample into Excel to create a chart.

    pH stability of the carrier


    Viability checking

    Preparation

    1. Prepare LB plates containing Amp.
    2. Prepare freeze-dried coated E. coli.
    3. Adjust PBS to pH 2.5 and pH 7.4 with HCl or NaOH.
    4. Prepare TB broth.

    Protocol

    1. Resuspend the freeze-dried E. coli powder with 1 mL of pH 2.5 PBS.
    2. Place the samples in a 37°C water bath for 1, 2, 4 hours.
    3. Centrifuge and remove the supernatant.
    4. Add 1 mL of pH7.4 PBS, centrifuge, and remove the supernatant to wash the remaining pH2.5 PBS.
    5. Resuspend the freeze-dried E. coli powder with 1 mL of pH 7.4 PBS.
    6. Place the samples in a 37°C water bath for 3 hours.
    7. Centrifuge and remove the supernatant.
    8. Add 1 mL TB into the samples to resuspend.
    9. Dilute the sample 500-fold. Take 50µL of samples and culture it on the LB plate for 18 hours.

    Growth behavior

    Preparation

    1. Prepare freeze-dried coated E. coli.
    2. Adjust PBS to pH 2.5 and pH 7.0 with HCl or NaOH.

    Protocol

    1. Resuspend the freeze-dried E. coli powder with 1 mL of pH 2.5 and pH 7.0 PBS.
    2. Place the samples in a 37°C water bath for 1 hour.
    3. Centrifuge and remove the supernatant.
    4. Add 1mL TB into the samples to resuspend.
    5. Check the O.D. 600 value.
    6. Dilute the samples until the O.D. values are 0.05 by adding TB.
    7. Add two glass beads to all test tubes containing samples and incubate at 37°C
    8. Measure the O.D. 600 value every 1 hour for 16 hours.
    9. Plot the data into a graph to see the growth behavior of E. coli under pH2.5 and pH7.0.

    Application-related experiments

    Sodium Caseinate Film

    1. Add 1 g of sodium caseinate to 40 mL of deionized water and stir at 360 rpm for 30 minutes.
    2. Add 0.25 g of glycerol and stir at 360 rpm for another 30 minutes.
    3. Place the mixture in a 50°C water bath and heat while shaking at 80 rpm for 15 minutes.
    4. Continue heating and shaking the solution at 50°C for 2 hours.
    5. Pour 40 mL of the solution into a petri dish with a diameter of 14 cm.
    6. Place the petri dish in an oven at 50°C until fully dried.
    7. Remove the film from the petri dish.

    Dissolution Rate Experiment 1

    1. While preparing the edible films mentioned above, add 0.3 g of crystal violet to every 100 ml of solution before proceeding with film formation.
    2. Measure 240 mL of deionized water, add the deep blue film containing crystal violet, and stir at 100 rpm.
    3. Every 20 minutes, withdraw 500 μL of the crystal violet film's water solution. Since the optimal tasting period for hand-shaken drinks is two hours, we set the test duration to end after two hours.
    4. For each time point (0 min, 20 min, 40 min, 60 min, 80 min, 100 min, 120 min), transfer 100 μL from the 500 μL samples to a 96-well plate with three replicates.
    5. Place the 96-well plate in a spectrophotometer to measure the absorbance at a wavelength of 570 nm.
    6. Use Excel to plot and analyze the dissolution of different films over time.

    Dissolution Rate Experiment 2

    1. Set up the equipment and dissolve the film in 240 ml of ddH2O.
    2. Immediately cover the container to ensure the flashlight is the only light source inside the equipment.
    3. After two hours of dissolution, import the current intensity data into MATLAB for analysis.

    Transparency Experiment

    1. Dissolve the transparent sodium caseinate film in 240 mL of commercially available unsweetened black tea.
    2. Stir at 100 rpm for 30 minutes, and withdraw 500 μL of the sodium caseinate film solution every 10 minutes.
    3. From the 500 μL samples taken at different times, withdraw 100 μL into a 96-well plate with three replicates.
    4. Place the 96-well plate in a spectrophotometer and measure the absorbance from 300 to 800 nm.
    5. Use Excel to plot and analyze whether the addition of sodium caseinate film affects the appearance and color of the commercially available unsweetened black tea.

    Reference


    1. William selleck, & Song tan. (2008). Recombinant Protein Complex Expression in E. Coli. https://doi.org/https://doi.org/10.1002%2F0471140864.ps0521s52
    2. Lin, huei-chih. (2018). Development of Active Edible Sodium Caseinate Packaging Films by Genipin Cross-Linking and Gallic Acid Incorporation.
    3. Khaoula khwaldia, Sylvie desobry-banon, Cristina perez-perez, & Stéphane desobry. (2004). Properties of Sodium Caseinate Film-Forming Dispersions and Films. https://doi.org/http://dx.doi.org/10.3168/jds.S0022-0302(04)70018-1
    4. Yujia qiu, Yan zhou, Yancheng chang, Xinyue laing, Hui zhang, Xiaorui lin, Ke qing, Xiaojie zhou, & Ziqiang lou. (2022). The Effects of Ventilation, Humidity, and Temperature on Bacterial Growth and Bacterial Genera Distribution. https://doi.org/http://dx.doi.org/10.3390/ijerph192215345
    5. Haoxiang wu, & Jonathan wong. (2022). Temperature versus Relative Humidity: Which Is More Important for Indoor Mold Prevention? https://doi.org/http://dx.doi.org/10.3390/jof8070696
    6. Xiaoming luo, Haixing song, Jing yang, Bin han, Ye feng, Yanbing leng, & Zhaoqiong chen. (2020). Encapsulation of Escherichia Coli Strain Nissle 1917 in a Chitosan―alginate Matrix by Combining Layer-by-Layer Assembly with CaCl2 Cross-Linking for an Effective Treatment of Inflammatory Bowel Diseases.