Record of Main Experimental Content
Pre-experiment safety training：
The main content is as follows：
Personal safety:
⦁ Wear laboratory coats.
⦁ Tie long hair up and secure loose clothing or accessories.
⦁ Always wear your mask and gloves before entering the laboratory.
⦁ Remember to wash hands before and after experiments.
Laboratory safety:
⦁ No eating or drinking.
⦁ Beware of chemical reactions and toxic substances.
⦁ In order to keep the laboratory tidy, avoid placing irrelevant items, such as notebooks, or waste materials.
⦁ After completing the experiment, turn off the electricity and close the doors and windows.
Biological safety:
⦁ Conduct all sterile procedures inside a laminar flow hood.
⦁ Laboratory waste must be disinfected.
Part 1: Amplification of target gene fragment by PCR
Purpose: Amplify DNA fragments by implement PCR Procedure.
Reagent Volume
PrimeSTAR Max Premix (2×) 10 μL
Forward Primer (10–15 pmol) ~0.2–0.3 μM
Reverse Primer (10–15 pmol) ~0.2–0.3 μM
Template 1 μL
ddH2O To 50 μL total
1.Prepare 20 μL PCR mixture.
1.95°C for 3 min
2.95°C for 30 s
3.53°C for 15 s
4.72°C for 1 min
5.72°C for 10 min
（Repeat step 2,3,4 for 33 times）
2.Run PCR as described above.
3.Analyze by agarose gel electrophoresis.
4.Visualize bands under UV light.
Participants: Lawrence Zeng, Dolores Yang, Jessica Shang, Noah Sun, Yuki Zhang, Iiya Wang, He Wang, James You, Lydia Zhu, Zhenze Xie, Dong Liang, Muhuan Guo
Part 2: Plasmid construction
Purpose:
Plasmids containing antimicrobial peptides and lysozyme were constructed by agarose gel electrophoresis, ligation after digestion, plasmid transformation and plasmid extraction.
Procedure:
1. Agarose Gel Preparation and Electrophoresis
1. Dissolve 0.1 g agarose in 100 mL TAE buffer by microwaving for 3 min.
2. Add 1 μL GelRed dye. Pour into mold with comb;
3. Place gel in electrophoresis chamber with TAE buffer.
4. Mix DNA samples with loading buffer, load into wells.
5. Run gel at 150 V for ~20 min.
6. Visualize bands under UV light.
2. DNA Gel Extraction and Purification
1. Check whether Washing Solution is adding anhydrous ethanol,
2. Check whether Buffer B2 appear precipitate,Set the water bath pot to 50℃
3. Cut out the gel block containing the target fragment from the agarose gel and weigh it.
4. Add Buffer B2 into the gel block and metal bath 55 ℃
5. Transfer sol solution into the adsorption column and 8000Xg centrifugation for 30 seconds
6. Add Wash Buffer and 9000Xg centrifugation for 30 seconds, pour the solution in the tube.
7. Repeat the previous step
8. Let empty adsorption column in the centrifuge for 1 minute
9. Put adsorption column into a clean 1.5mL centrifuge tube and add 30μl Elution Buffer into the center of the adsorption film. Stewing for 1 minute and centrifugation for 1 minute.
3. Transformation into Competent Cells
1. Preparation for antibiotic plates (LB+1:1000 bleomycin)
2. Thaw competent E. coli DH5α cells on the ice.
3. Add 10μL recombinant product (plasmid) into the competent cells, gently flick vessel wall, gently mix it by using the pipette tip. Incubate on ice for 30 minutes, stationary.
4. 42℃ hot water bath heat shocks for 45s in order to create channels and pores in the cell membrane that connect the cell interior to the external environment.
5. Remove immediately, incubate on ice for 2 minutes to reduce the temperature.
6. Add 900μL LB culture medium. Incubate at 37℃ with shaking at 200rmp for one hour.
7. Centrifuge and discard 800μL of the supernatant.
8. Separate the thallus in the remaining 200μL of LB medium.
9. Evenly spread the sample on antibiotic plates by using a sterile cell spreader.
4.Colony PCR
Reagent Volume
PrimeSTAR Max Premix (2×) 10 μL
Forward Primer (10–15 pmol) ~0.2–0.3 μM
Reverse Primer (10–15 pmol) ~0.2–0.3 μM
Template (colonies) 1 μL
ddH2O To 50 μL total
1.Prepare 20 μL PCR mixture.
1.95°C for 3 min
2.95°C for 30 s
3.53°C for 15 s
4.72°C for 1 min
5.72°C for 10 min
（Repeat step 2,3,4 for 33 times）
2.Run PCR as described above.
3.Analyze by agarose gel electrophoresis.
4.Visualize bands under UV light.
Part 3 SDS-PAGE was used to verify whether the protein was expressed
Purpose:
Verify whether antimicrobial peptides and lysozyme are successfully expressed.
Procedures:
1. Protein Extraction
⦁ Add lysozyme and lysis buffer to bacterial pellet. Mix until fully resuspended.
⦁ Place tube in an insulated container with ice. Sonicate at 50% power (200–300 W) with 5-second intervals, total of 6 minutes.
⦁ Centrifuge lysate at 1000 × g for 10 seconds at 4°C. Collect supernatant (crude protein extract).
⦁ Prepare column in parallel by resuspending nickel affinity gel and equilibrating it with lysis buffer at 4°C.
2. Protein Purification
⦁ Mix crude extract with equilibrated resin. Shake at 4°C for 1.5 hours.
⦁ Load mixture into gravity column. Let drain slowly and repeat 3 times. Backup samples.
⦁ Wash with washing buffer and collect flow-through (wash fractions).
⦁ Elute protein with elution buffer. Collect elution fractions and label clearly.
3. SDS-PAGE Validation
Part 4 The growth curve of Pichia pastoris
Purpose:
The growth curve measurement was used to verify whether the growth ability of yeast was different when different genes were introduced.
Procedures:
Inoculate the transformants into 3 mL of YPD liquid medium and add bleomycin
Zecoin, then incubate at 30°C (OD600=0.02), The growth level of the strain was determined at a fixed time and the growth curve was drawn with the same
absorbance..
Part 5 Determination of Minimum Inhibitory Concentrations of antimicrobial peptides and lysozyme
Purpose:
Verify the bactericidal activity of antimicrobial peptides and lysozyme.
Procedures:
Prepare 1.2ml solutions of antimicrobial peptides in a centrifuge tube1(128 μg/mL)
Transfer 600μl solutions of antimicrobial peptides from tube1 into the centrifuge tube2, then add 600μl sterile water.
Transfer 600μl solutions of antimicrobial peptides from tube 2 into the centrifuge tube3, then add 600μl sterile water.
Transfer 600μl solutions of antimicrobial peptides from tube 3 into the centrifuge tube4, then add 600μl sterile water.
Transfer 600μl solutions of antimicrobial peptides from tube 4 into the centrifuge tube5, then add 600μl sterile water.
Transfer 600μl solutions of antimicrobial peptides from tube 5 into the centrifuge tube6, then add 600μl sterile water.
Transfer 600μl solutions of antimicrobial peptides from tube 6 into the centrifuge tube7, then add 600μl sterile water.
Transfer 600μl solutions of antimicrobial peptides from tube 7 into the centrifuge tube8, then add 600μl sterile water.
All antimicrobial peptides and lysozyme were diluted in gradient according to the above scheme.
Application of each sample into 96-well bacterial culture plate in order
Application of bacterial into the first line of 96-well bacterial culture plate
Application of ampicillin into the second line of 96-well bacterial culture plate
Application of LB culture medium into the third line of 96-well bacterial culture plate
Cultivate the bacterial culture until the OD value reaches 0.9–1.0 (with a CFU count of 10⁹ CFU/mL).
Perform a 1000-fold dilution to adjust the CFU count to 10⁶ CFU/mL.
Application diluted bacterial into 96-well bacterial culture plate.
Part 6 Determination of Minimum Bactericidal Concentration of antimicrobial peptides and lysozyme
Purpose:
Verify the bactericidal activity of antimicrobial peptides and lysozyme.
Procedure:
We measured this by applying the targeted proteins and the bacteria
(E.coli or Bacillus subtilis) onto petri dishes with LB culture medium.
Use the protein solutions we made, dilute them to two separate centrifugal tubes
(128 μg/ml and 64 μg/ml).
For each type of targeted protein, we created 4 petri dishes LB culture medium.
Each for:
a. 128μg/ml concentration of protein + E. coli
b. 64μg/ml concentration of protein + E. coli
c. 128μg/ml concentration of protein + Bacillus subtilis
d. 64μg/ml concentration of protein + Bacillus subtilis
Add 100μl of protein to each petri dish, then use disposal cell
Use spreader to spread the solution evenly across the culture medium.
Use parallel pipette to add 100μl of bacteria solution to each corresponding petri dish,
then use disposal cell spreader to spread the solution evenly across the culturemedium.
Put the petri dishes into the constant-temperature incubator for 24 hours.
Use the microplate reader to visualize the results.
Part 7 Time-Kill curve of antimicrobial peptides and lysozyme
Purpose:
Verify the bactericidal activity of antimicrobial peptides and lysozyme.
1.Add the Bacillus subtilis and Escherichia coli separately into our antibacterial
peptides and lysozymes, into a EP tube, using 500 μL of each component.
2. Add the sample into the 96 cell culture plate, starting from hour 0.
3. Samples were collected every 2 hours and OD600 was determined.
Part8 Laser Scanning Confocal Microscope observation
Purpose: The bactericidal effect of antimicrobial peptides and lysozyme was determined by living dead cell staining.
Escherichia coli and Bacillus subtilis were treated with antimicrobial peptides and lysozyme at 37 for 24 hours, respectively.
1.The cell precipitate obtained by centrifugation was resuspended with 1XAssay Bufer, so that the cell count of the resuspended cell suspension was 1X105-6 cells /ml.
2.Add 1-2ul of Calcein-AM (stock solution) per 1ml of cell volume, blow and mix evenly, and incubate at 37℃ in the dark for 20-25 min.
3.Add 3-5ul of PI stock solution from the kit into the stained cells, and dye it in the dark at room temperature for 5min.
4.Incubate the fluorescent cells for 5min minutes with 450g, and remove the staining solution by centrifugation.
Note: It is suggested that cells should be kept away from light during fluorescent staining.
5.Wash 450g cells with 1x PBS for 5min, resuspend the cells with 1x PBS after centrifugation, take 3-5ul drops on a clean glass slide, and after tabletting with a clean cover glass, please examine them under fluorescence microscope in time. (6) Using 490±10 nm excitation filter to simultaneously detect living cells (yellow-green fluorescence) and dead cells (red fluorescence) under fluorescence microscope. In addition, only dead cells can be observed by using 545nm emission filter. It can also be detected directly by using a suitable filter under a fluorescent enzyme-labeled instrument.
E.coli
Part9 Morphological observation of scanning electron microscope
Purpose: The morphology of bacteria treated with antimicrobial peptides and lysozyme was observed by scanning electron microscope.
1. Sampling: Generally, the sample size is not more than 10x10 x 5mm. Determine the target of observing the sample to avoid pulling and squeezing the sample during sampling.
2. Cleaning: remove impurities from the observation surface and fully expose the observation surface without damaging it.
3. Fixation: Generally, it is better to fix glutaraldehyde and acid at 4℃.
Glutaraldehyde and acid can be fixed separately or double-fixed with "glutaraldehyde-osmium acid". After fixing for 1-3 hours, the fixed solution is sucked out, 0,1M of cool acid buffer wave (PH=7.2) is added, and the solution is rinsed for 3-4 times for 1 hour.
4. Dehydration: Suck out the buffer wave from the vial, add ethanol to dehydrate step by step (the ethanol concentration is 30%-50%-70%6-80%-90%-100% at a time), and the dehydration time depends on the sample size, generally 15-30 minutes. Add the 1:1 mixture of isoamyl acetate and ethanol, soak for 10-20min and shake properly, discard the mixture, add pure isoamyl acetate and soak for 10-20min and shake properly.
5. Drying: Drying is a key link in the biological preparation of scanning electron microscope. It is necessary to reduce the distortion of surface morphology caused by water evaporation surface as much as possible during the drying process, and it is necessary to ensure thorough drying. Commonly used methods include natural drying, dry drying, critical point drying, freeze drying and vacuum drying. Different drying methods will be selected for different biological samples.
6. Sample loading: the surface is plated with gold and observed by scanning electron microscope.
