Experiment

Experiment

Table of Contents

I. Bacterial Growth Curve Experiment

Purpose

1. L. lactis: To determine how long L. lactis grows before entering the stationary phase. This information will help estimate how often PANDAGE needs to be replaced and can also be used for calculating the production rate of growth factors.

2. E. coli: To establish a bacterial growth model after inoculation but before drug treatment. This will serve as a reference for detection and drug administration, helping us predict the optimal timing for treatment. Additionally, the time vs. concentration curve will assist in preparing appropriate bacterial concentrations for other experiments.

Materials & Equipment

Purpose

  • MRS broth
  • Sterile flasks
  • Spectrophotometer
  • Incubator (37°C for E. coli, room temperature for L. lactis)
  • Pipettes and tips: For transferring bacterial cultures and media.
  • Sterile water: To prepare dilutions and rinse equipment if needed.
  • Data recording tools: For logging OD readings and time points throughout the experiment.

Protocol

  1. Incubate bacterial culture tubes in a shaking incubator at 37°C for E. coli and at room temperature for L. lactis.
  2. At regular intervals (every 2 hours), vortex the culture tubes and measure the OD. Then, extract 0.1 mL of the culture for CFU counting.

II. CFU Assay

Purpose

By inferring the number of viable bacteria in each time point’s culture tube from the viable cell counts obtained after diluting the bacterial solution to a small volume.

Materials

LB plate (without antibiotic)、bacterial solution、ddH2O、pipetman / tips / Eppendorf

Protocol

  1. Divide the LB plate into four sections and label the plate.
  2. Aspirate the bacterial solution from the culture tube into an Eppendorf tube and perform a 10-fold dilution each time (0.1 mL of the previous tube’s bacterial solution + 0.9 mL of dd water).
  3. Use the diluted bacterial solution in the Eppendorf tube to spot onto the corresponding labeled area of the LB plate using the spotting method (5 μL per spot, with five spots per section).
  4. When the colonies have grown to an appropriate size, place the LB plate under an inverted phase-contrast microscope to count the colonies.
  1. Pipette 100 µL of DH5-alpha E. coli from the stock and then aliquot 50 µL into another eppendorf tube. Place the tube on ice.
  2. Add 5 µL of ligation product to the aliquoted DH5-alpha E. coli, flick the tube, and incubate it on ice for 25 minutes, flicking gently every ten minutes.
  3. Place both tubes in a 42°C dry bath for 45 seconds.
  4. In a laminar flow hood, add 1 ml of LB medium with ampicillin to each tube, and incubate them at 37°C with shaking for 1 hour.
  5. Take 100 µL of the mixture and spread it evenly on a LB medium plate with ampicillin using a disposable cell spreaders.
  6. Incubate the LB medium plate with ampicillin in an incubator at 37°C for 16 hours.

III. Cloning

A. Ligation

Materials & Equipment

  1. 2X Reaction Buffer (Thermo Fisher Scientific)
  2. pJET1.2/blunt Cloning Vector (50 ng/µL) (Thermo Fisher Scientific)
  3. T4 DNA Ligase (400,000 U/mL) (New England Biolabs)
  4. Sample (insert)
  5. Nuclease-free water (Protech Technology)
  6. Dry bath
    • Protocol
    1. Set up the ligation reaction on ice. Vortex briefly and centrifuge for 3-5 s.
    2. Incubate the ligation mixture at room temperature (22 °C) for 30 min.
    3. Use the ligation mixture directly for transformation.

B. Transformation

Materials & Equipment

  1. Dry bath
  2. Disposable cell spreaders
  3. Incubator
  4. DH5-alpha E. coli (New England Biolabs)
  5. Sample (ligation product)
  6. LB medium plate with ampicillin
  7. LB medium with ampicillin (0.1 mg ampicillin / 1 mL LB)

Protocol

  1. Pipette 100 µL of DH5-alpha E. coli from the stock and then aliquot 50 µL into another eppendorf tube. Place the tube on ice.
  2. Add 5 µL of ligation product to the aliquoted DH5-alpha E. coli, flick the tube, and incubate it on ice for 25 minutes, flicking gently every ten minutes.
  3. Place both tubes in a 42°C dry bath for 45 seconds.
  4. In a laminar flow hood, add 1 ml of LB medium with ampicillin to each tube, and incubate them at 37°C with shaking for 1 hour.
  5. Take 100 µL of the mixture and spread it evenly on a LB medium plate with ampicillin using a disposable cell spreaders.
  6. Incubate the LB medium plate with ampicillin in an incubator at 37°C for 16 hours.

C. Plasmid extraction

Materials & Equipment

  1. Dry bath
  2. Centrifuge
  3. 50% Glycerol
  4. Mini Plus Plasmid DNA Extraction System (Viogene)
  5. Sample (colony PCR product)

Protocol

  1. Take 500 µL of a 16-hour bacterial culture and use it for bacterial cryopreservation. Mix the 500 µL bacterial culture with 500 µL of glycerol in an Eppendorf tube, then store it at -80 °C.
  2. Take 1 mL of the remaining 4.5 mL bacterial culture at a time and centrifuge it 1 min at 10,000rpm in Eppendorf tubes. After each centrifugation, discard the supernatant using a pipette.
  3. Add 200 µL Solution1 and pipette it until the cells are completely resuspended.
  4. Add 200 µL Solution2 and mix by gently inverting the capped tube 5-6 times. Incubate at room temperature for 5 min.
  5. Add 300 µL Solution3 and mix by gently inverting the capped tube 5-6 times. Incubate at room temperature for 3 min.
  6. Centrifuge it at 10,000 rpm for 5 minutes. You will observe the formation of a dense, white pellet either along the side or at the base of the tube.
  7. Load the spin column into a collection tube, transfer the supernatant from step 6 directly to spin column, spin for 1 min at 10,000 rpm.
  8. Take out the spin column from the collection tube, dispose of the liquid that flows through it, reinsert the spin column, and then apply 700 µL of washing solution. Centrifuge for 1 minute.
  9. Take out the spin column from the collection tube, pour away the liquid, reinsert the spin column, and then apply 700 µL of washing solution. Centrifuge for 1 minute.
  10. Dispose of the filtrate and then centrifuge at 10,000 rpm for 3 minutes to eliminate any remaining traces of ethanol. Incubate the spin column in a 60 °C oven for 8 minutes.
  11. Remove the spin column and place the column in a new Eppendorf tube.
  12. Add 50 µL free water into the column. Elute DNA by centrifugation for 1 min and store the eluted DNA at -20 °C.

D. DNA Electrophoresis

Materials & Equipment

Pipettes and tips、Agarose gel、Gel box、Agarose powder、TAE buffer、DNA marker、Loading dye、Ethidium bromide 、UV transilluminator、Microwave

Protocol

  1. Dissolve agarose in TAE buffer by heating it in a microwave or hotplate. Cool slightly and pour it into a gel tray with a comb to create wells.
  2. Once the gel is solidified, place it in the gel box and submerge it in electrophoresis buffer.
  3. Add loading dye to the DNA samples and carefully pipette the samples into the wells. Also load the DNA ladder as a reference.
  4. Attach the gel box to the power supply, ensuring the DNA runs towards the positive electrode (anode). Run at 80-120V for 30-60 minutes, depending on the size of the gel and DNA fragments.
  5. If necessary, stain the gel using ethidium bromide or another DNA stain to visualize the DNA.
  6. Place the gel on a UV transilluminator to view the separated DNA bands.

IV. Hydrogel Preparation

  1. Prepare 50 mM acetic acid with 1% v/v glutaraldehyde.
  2. Add 1 g of chitosan to 50 mM acetic acid (2% w/v) to prepare the chitosan solution（stir overnight）, and adjust the pH to 6.3-6.5.
  3. Centrifuge the precipitate at 1000 rpm for 1 minute.
  4. Mix the chitosan solution with 1% v/v glutaraldehyde crosslinking agent at a 5:1 ratio.
  5. Let it stand for 3-5 minutes to allow gel formation.

V. CS Hydrogel Preparation

  1. 300 mg of CS was dissolved in 1.14 ml of 1 M NaOH in 15 ml tube and mix well with a stirring rod.
  2. 279 ul of BDDE cross-linker was added and mixed thoroughly with a voretx at 1000 rpm for 30 minutes.
  3. Use pipetteman to transfer the ungelatinized thick liquid to a 6 cm dish.
  4. Use tape to fix the dish on the test tube rack, and put it in the water bath under 60℃ for 60 minutes. ( Make sure the water level is higher than the height of the thick liquid.)

VI. AMP inhibition test

  1. Culture the bacterial cells until the OD600 reaches 0.5.
  2. Dilute the bacterial culture with LB broth to a final concentration of 5 × 10^5 cells/mL.
  3. Adjust the concentration with ddH2O as needed to prepare for colony-forming unit (CFU) analysis.
  4. Divide the LB agar plate into four sections and appropriately label each section.
  5. Transfer the bacterial solution from the culture tube into an Eppendorf tube and perform serial 10-fold dilutions (add 0.1 mL of the bacterial solution to 0.9 mL of ddH2O in each subsequent tube).
  6. Using the spotting method, apply 5 μL of the diluted bacterial solution from each Eppendorf tube to the corresponding labeled section on the LB plate, spotting five replicates per section.
  7. Once the bacterial colonies have reached an appropriate size, count the colonies using an inverted phase-contrast microscope.

VII. SDS-PAGE

Making Gel

Lower gel of WB   10mL Upper gel of WB   3mL
15% H2O 2.3 5% H2O 1.68
  30% acrylamide 5.0   30% acrylamide 0.51
  1.5M Tris(pH8.8) 2.5   1.5M Tris(pH8.8) 0.75
  10% SDS 0.1   10% SDS 0.03
  10% APS 0.1   10% APS 0.03
  TEMED 0.004   TEMED 0.003
  • ddH2O should be added first to prevent precipitation. TEMED must be added the last because it will cause the solution to condense.
  1. Add the lower gel to the lower edge of the green frame, and use isopropanol to press the horizontal line. When the lower gel is formed, pour out the isopropanol, add the upper gel to the drowning out, and insert the well combs.

Loading Samples and Running Gel

  1. Sample：Loading buffer = 4：1
  2. Heat at 95℃ dry bath for 10 minutes. (Eppendorf with Cap Holding Tabs.)
  3. Centrifuge 13000rpm for 1 minute.
  4. Set up the apparatus and make sure it is not leaking by adding ddH2O into it.
  5. 1X Running buffer fill up the middle of the apparatus.
  6. Load Marker into the first and the last lane of the gel(first lane: 5 µl, last lane: 2 µl), then load 25 ul samples into the additional wells.
  7. Fill the apparatus with 1X Running buffer until the wire is covered.
  8. First, run the upper gel at 55V for 30 minutes, then run the lower gel at 110V for 60 minutes. (Or run both gels at 300V, 80-90A, 30 minutes.)

Dyeing Gel

Staining Solution (CBR Staining Solution):

Dissolve 1 mg of Coomassie Brilliant Blue R-250 in 250 mL of water, then add 250 mL of methanol and 50 mL of acetic acid. Mix thoroughly to completely dissolve the CBR.

Destaining Solution (20% Methanol and 10% Acetic Acid):

In a 1L graduated cylinder, first add 200 mL of methanol and 100 mL of acetic acid. Then, fill with water to make a total volume of 1L. Mix well and store in a sealed container for later use.

  1. Take out the gel assembly, gently pry up the glass, and remove the gel. Cut off any excess parts that do not need to be stained.
  2. Take a staining tray (slightly larger than the gel), pour in the CBR staining solution, and use gravity to let the gel fall into the staining tray.
  3. Stain the gel in the CBR solution for about 30 minutes. Make sure the staining tray is covered and place it on a slow rotating platform (50 rpm). Ensure that the gel is fully submerged in the staining solution, as incomplete submersion will cause uneven staining.
  4. After staining, carefully pour the CBR staining solution back into its original bottle. The entire gel should now be blue. Rinse off any remaining staining solution with tap water, then pour about 20 mL of destaining solution into the tray. Cover the tray, place it back on the rotating platform, and continue shaking. (Note: Wear gloves during the staining and destaining processes to avoid leaving fingerprints on the gel.)
  5. The blue background of the gel will gradually fade. When the destaining solution turns blue, replace it with fresh destaining solution. Repeat this destaining process several times, and within an hour, blue protein bands should become visible. Used destaining solution should be collected and disposed of in an organic solvent waste container.
  6. Once the gel background becomes mostly transparent, the staining and destaining process is complete. Pour off the destaining solution, wash the gel once or twice with 50% methanol solution, and allow the gel to shrink to approximately its original size. It can then be prepared for drying.

VIII. ELISA

  1. prepare the GM-CSF standard samples to create a standard curve. The setting volume and concentration are at right.
  2. prepare the sample.
  3. calculate the number of wells needed for the assay. Return any extra wells to the aluminum foil bag with a desiccant packet, and allow the wells to equilibrate to room temperature before use.
  4. add 100 μL of diluted samples (within the detection range) to the wells of a microplate. Cover the plate with a lid and incubate at room temperature for 2 hours.
  5. fill the wells with wash buffer and let them sit for 1-2 minutes, repeat for three times. After the final wash, invert the plate and tap it on paper towels to dry.(use a multi-channel pipette or an automated microplate washer)
  6. add 100 μL of hGM-CSF detection antibody to each well. Cover the plate and incubate at room temperature for 1 hour.
  7. repeat the washing procedure described in step 5 three times.
  8. add 100 μL of 1X Streptavidin-HRP to each well. Cover the plate and incubate at room temperature for 30 minutes. (avoiding direct sunlight)
  9. repeat the washing procedure described in step 5 five times.
  10. add 100 μL of HRP substrate (TMB) to each well. Cover the plate and incubate at room temperature for 15 minutes. (avoiding light)
  11. add the stop solution to each well. The color in the wells should change from blue to yellow. If the color is green or appears uneven, gently tap the plate to mix thoroughly.
  12. within 30 minutes, read the absorbance of each well at 450 nm using a microplate reader.
  13. repeat the readings and calculate the average value. Subtract the OD value of the zero standard (1-Std.8) from all readings to correct for background. Record all data.

IX. Aptamer Reduction and Au Conjugation

Preparing 10X aptamer solution

  1. Briefly centrifuge the aptamer tube (ensure the dried aptamer pellet is at the bottom)
  2. Adding 200 ul ddWater to resuspend the DNA aptamer (10X working concentration)
  3. Quickly spin the sample down (>1000 x g for a few seconds)
  4. Aliquoting and storing in a non-defrosting -20°C freezer.

Reduction for Thiol-modified aptamers

  1. treating the aptamer with TCEP (Tris [2-carboxyethyl] phosphine), usually add TCEP in 100X excess (30mM TCEP to 300 uM aptamer)
  2. keeping for 2 hours at room temperature to reduce the aptamer
  3. no need to remove the TCEP from aptamer before using it in assays.

Preparing working aptamer

  1. adding 50 mM Tris-acetate (PH 8.2) making aptamer solution to working concentration
  2. heating the solution at 95°C for 5 min
  3. incubating for 15 min at room temperature
  4. aptamers are conformationally stable for at least one week at room temperature

Incubating aptamer on Electrode pad

  1. adding 5 ul aptamer solution of 5uM on Electrode pad
  2. incubating for 16 hours at room temperature
  3. all step need to do in hood and clear plate to transport

X. Hydrogel Diffusion

  1. Add chitosan hydrogel and crosslinking agent in a 5:1 ratio to an Eppendorf tube until reaching a total volume of 1 mL.
  2. Add 10 µL of GM-CSF solution to the center.
  3. In another Eppendorf tube, add the hydrogel and crosslinking agent in a 5:1 ratio.
  4. Gently pipette the mixture (avoiding bubble formation).
  5. Quickly aspirate 200 µL and add it to the Eppendorf tube containing GM-CSF, covering the top.
  6. Add 200 µL of PBS.
  7. When collecting the sample, gently mix the liquid by pipetting, and aspirate 50 µL (without damaging the hydrogel).
  8. Store the sample at -20°C.
  9. Perform an ELISA test.

XI. Cell Culture

  1. Objective: To culture TF-1 cells for subsequent experiments and to ensure their healthy growth.

    1. The initial medium preparation involved first dissolving BSA in PBS, filtering it, and preparing a 0.1% BSA in PBS solution. Next, we dissolved GM-CSF in 0.1% BSA at a concentration of 100 μg/mL, aliquoting the GM-CSF solution into 2 µL portions and storing them at -20°C. One aliquot of GM-CSF was mixed with medium and FBS to prepare an RPMI-1640 medium containing 2 ng/mL GM-CSF, 1% ampicillin and 10% FBS. Finally, the TF-1 cells were thawed from liquid nitrogen, resuspended in 9 mL of medium, centrifuged, and seeded into T25 flasks, after which the results were observed under a microscope.
    2. According to the manufacturer’s description, TF-1 cells tend to be unstable during culture, which may result in difficulties adhering to the flask. During the first generation of cell culture, we did observe some cells floating, but the majority of the cells appeared to be in a healthy condition.
    3. In subsequent passages, if the medium turned yellow, we transferred the floating TF-1 cells to four new T25 flasks and passaged the adherent cells. We observed that only a small portion of the cells adhered to the flask, though not a sufficient number. We also performed a trypan blue exclusion test to confirm cell viability, and the results indicated that the cells were alive.
    4. However, in later passages, we noticed an increasing number of floating cells, and no improvement was observed regardless of how frequently we changed the medium. Additionally, white, sand-like impurities appeared in the T25 flasks, leading us to suspect contamination during the passaging process. We cultured RPMI-1640, RPMI-1640 + GM-CSF, and RPMI-1640 + GM-CSF + FBS in bacterial culture tubes overnight to check for contamination. The results confirmed that bacterial contamination likely occurred due to improper handling during the cell passaging. After discussion, we decided that ampicillin should be added to the medium to prevent this issue from recurring.
    5. We restarted the cell culture, ensuring that the medium contained RPMI-1640 + GM-CSF + FBS + ampicillin. After several successful passages, the cells were able to survive. However, due to time constraints, we have not yet been able to use the TF-1 cells to test the secretion and bioactivity of the GM-CSF we transfected, which will be part of our future work.