Preparation of culture medium
Goal:Prepare a culture environment with rich nutrients for bacterial growth
Materials:
Tryptone
Yeast Extract
ddH2O
Pipette and tips
PCR tube
EP tube
Peptone
K2HPO4
KH2PO4
Gloves
Lab coat
Procedure:
LB Medium: Weigh 2.5 g tryptone, 1.25 g yeast extract, 2.5 g NaCl. Add distilled water to 250 mL. Sterilize at 121°C for 30 min. For solid medium, add 2% agar powder before sterilization.
YPD Medium: Weigh 2.5 g yeast extract, 5 g tryptone. Prepare glucose stock separately, sterilize separately. Add water to 250 mL and autoclave at 121°C for 20 min. Add glucose before use. Add 2% agar powder for solid medium.
BMGY Medium:, Weigh 0.5 g yeast extract, 1g peptone,0.15g K2HPO4
0.59g KH2PO4, Dissolve with 50ml of water. Sterilize at 121°C for 20 min. (5 mL 10×YNB, 100 μ l of 50× biotin and 500μL of glycerol were added during fermentation.)
BMMY Medium:Weigh 0.5 g yeast extract, 1g peptone,0.15g K2HPO4,0.59g KH2PO4, Dissolve with 50ml of water. Sterilize at 121°C for 20 min. (During fermentation, 5ml of 10× YNB, 100μ l of 50× biotin and 0.5 g of methanol were added.)
The Construction of Plasmid
⦁ Construction of pPICZα A/hLYZ
Goal: Construction of plasmid containing lysozyme hLYZ
A: Obtain the target gene
Materials:
Template DNA
PrimeSTAR Max Premix
Forward Primer (10 μM)
Reverse Primer (10 μM)
ddH2O
Pipette and tips
PCR tube
EP tube
Agarose
TAE Buffer
GelRed
DNA Loading Buffer
DNA marker
Procedure:
1.Dispense template DNA into tubes.
2.Add corresponding forward and reverse primers.
3.Add PrimeSTAR Max Premix and ddH2O to each tube.
4.Mix gently and run PCR using:
95˚ 3 minutes
one cycle:
95˚for 30 seconds
55˚for 30 seconds
72˚ for 50 seconds
This cycle repeats for 33 times
72˚ for 10 minutes
B: Agarose Gel Electrophoresis
Goal: Verify whether the target gene is constructed correctly
Materials:
Agarose
TAE Buffer
GelRed
DNA Loading Buffer
DNA marker
Procedure:
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.
C: DNA Gel Extraction
Goal: Obtaining DNA fragments with correct construction.
Materials:
Binding Buffer
Wash Buffer
Elution Buffer
Procedure:
⦁ Check whether Washing Solution is adding anhydrous ethanol,
⦁ Check whether Buffer B2 appear precipitate,Set the water bath pot to 50℃
⦁ Cut out the gel block containing the target fragment from the agarose gel and weigh it.
⦁ Add Buffer B2 into the gel block and metal bath 55 ℃
⦁ Transfer sol solution into the adsorption column and 8000Xg centrifugation for 30 seconds
⦁ Add Wash Buffer and 9000Xg centrifugation for 30 seconds, pour the solution in the tube.
⦁ Repeat the previous step
⦁ Let empty adsorption column in the centrifuge for 1 minute
⦁ 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.
D: Restriction enzyme digestion and ligation
Goal: Obtaining DNA fragments with restriction sites.
Materials:
Plasmid DNA
Restriction enzyme 1
Restriction enzyme 2
10× Reaction Buffer
Nuclease-free Water
Ligation buffer
T4 ligase
Insert DNA fragment
Digested vector
ddH2O
Procedure:
1.Mix all components in a 30 μL total reaction volume.
2.Incubate at 37°C for 3 hours.
3.Mix insert fragmen(step 2)t with ligation buffer, T4 ligase, digested vector,
And ddH2O.
4.Incubate at room temp for 1 hour.
E: Transformation into E.coli Competent Cells
Goal: Expression of plasmid in Escherichia coli.
Materials:
Competent E. coli cells
LB culture medium
Peptone, yeast extract, NaCl (for making LB agar)
Kanamycin
Petri dishes
Sterile cell spreader
Transformed bacterial colony plates
Procedure:
⦁ Preparation for antibiotic plates (LB+1:1000 bleomycin)
⦁ Thaw competent E. coli DH5α cells on the ice.
⦁ 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.
⦁ 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.
⦁ Remove immediately, incubate on ice for 2 minutes to reduce the temperature.
⦁ Add 900μL LB culture medium. Incubate at 37℃ with shaking at 200rmp for one hour.
⦁ Centrifuge and discard 800μL of the supernatant.
⦁ Separate the thallus in the remaining 200μL of LB medium.
⦁ Evenly spread the sample on antibiotic plates by using a sterile cell spreader.
F: Colony PCR
Goal: Verification whether the strain is constructed correctly.
Materials:
PrimeSTAR Max Premix (2×)
Forward Primer (10–15 pmol)
Reverse Primer (10–15 pmol)
ddH2O
Template (colonies)
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 (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.
G: Plasmid Extraction
Goal: Obtain the successfully constructed plasmid.
Materials: Plasmid extraction kit
Procedure:
Cell Lysis:
1.Harvest 1.5–5 mL overnight bacterial culture; centrifuge at 8,000 × g for 2 min; discard supernatant.
2.Resuspend pellet in 250 μL Buffer SP1; mix thoroughly.
3.Add 250 μL Buffer SP2; mix gently by inversion 5–10 times; incubate 2–4 min at
room temperature.
4.Add 350 μL Buffer SP3; mix gently by inversion 5–10 times
Binding and Washing:
1.Centrifuge at 12,000 × g for 5–10 min; transfer lysate to spin column; centrifuge at 8,000 × g for 30 sec; discard flow-through.
2.(Optional) Add 500 μL Buffer DW1; centrifuge at 9,000 × g for 30 sec; discard flow-through.
3.Add 500 μL Wash Solution; centrifuge at 9,000 × g for 30 sec; discard flow-through.
4.Repeat wash step once.
5.Dry spin column at 9,000 × g for 1 min.
Elution:
1.Place spin column in a clean 1.5 mL tube.
2.Add 50–100 μL Elution Buffer to membrane center; incubate 1 min; centrifuge for 1 min to elute DNA.
H: Transforming plasmid into Pichia pastoris.
Goal: Make lysozyme protein express in Pichia pastoris.
Materials:
GS115 Chemically Competent Cell
Yeast transformation promoting reagent
Pichia pastoris transformation solution
Procedure:
1. Take 5μg of plasmid to be transformed, cut by enzyme and recover;
2. Take a sterile 1.5 ml EP tube, add 1-5ug of precooled linear plasmid (the volume is not higher than 15 μL), 5 μL of yeast transformation promoter, 100 μL of GS115 competent cells melted on ice and 500 ml of Pichia pastoris transformation solution in turn, gently turn and mix evenly for 6-8 times.
3.Water bath at 30°C for 30min, gently turn over and mix evenly for 6-8 times every 15min.
4. Add 20 mL of dimethyl sulfoxide to each branch (to improve the conversion efficiency, it is not necessary).
5.Take a water bath at 42°C for 15min, gently turn over and mix evenly for 6-8 times every 7.5min.
6.The supernatant was instantly centrifuged at 12000rpm, and each YPG Plus (or YPG) with lml was resuscitated at 30℃ for 1 hour (to improve the transformation efficiency, it was not necessary).
7.The supernatant was instantly centrifuged at 12000rpm, resuspended with 100μ 0.9% NaCl, smeared and cultured at 30℃ for 3-7 days.
8. Verify whether the strain is constructed correctly by colony PCR.
⦁ Construction of pPICZα A/Cecropin A
A: Obtain the target gene
Goal: Obtaining a gene fragment for constructing antibacterial peptide Cecropin A
Materials: The specific primers of antimicrobial peptide A were designed,
and the other materials were the same as those in 2.1-A.
Procedure: The procedure is basically the same as 2.1-A, and the
PCR extension time is adjusted according to the fragment length.
B: Agarose Gel Electrophoresis
Goal: Verify whether the target gene is constructed correctly
Materials: Materials were the same as those in 2.1-B.
Procedure: The procedure is basically the same as 2.1-B.
C: DNA Gel Extraction
Goal: Obtaining DNA fragments with correct construction.
Materials: Materials were the same as those in 2.1-C.
Procedure: The procedure is basically the same as 2.1-C.
D: Restriction enzyme digestion and ligation
Goal: Obtaining DNA fragments with restriction sites.
Materials: Materials were the same as those in 2.1-D.
Procedure: The procedure is basically the same as 2.1-D.
E: Transformation into E.coli Competent Cells
Goal: Expression of plasmid in Escherichia coli.
Materials: Materials were the same as those in 2.1-E.
Procedure: The procedure is basically the same as 2.1-E.
F: Colony PCR
Goal: Verification whether the strain is constructed correctly.
Materials: Materials were the same as those in 2.1-F.
Procedure: The procedure is basically the same as 2.1-F.
G: Plasmid Extraction
Goal: Obtain the successfully constructed plasmid.
Materials: Materials were the same as those in 2.1-G.
Procedure: The procedure is basically the same as 2.1-G.
H: Transforming plasmid into Pichia pastoris.
Goal: Make lysozyme protein express in Pichia pastoris.
Materials: Materials were the same as those in 2.1-H.
Procedure: The procedure is basically the same as 2.1-H.
⦁ Construction of pPICZα A/Melittin
A: Obtain the target gene
Goal: Obtaining a gene fragment for constructing antibacterial peptide Melittin
Materials: The specific primers of antimicrobial peptide A were designed,
and the other materials were the same as those in 2.1-A.
Procedure: The procedure is basically the same as 2.1-A, and the
PCR extension time is adjusted according to the fragment length.
B: Agarose Gel Electrophoresis
Goal: Verify whether the target gene is constructed correctly
Materials: Materials were the same as those in 2.1-B.
Procedure: The procedure is basically the same as 2.1-B.
C: DNA Gel Extraction
Goal: Obtaining DNA fragments with correct construction.
Materials: Materials were the same as those in 2.1-C.
Procedure: The procedure is basically the same as 2.1-C.
D: Restriction enzyme digestion and ligation
Goal: Obtaining DNA fragments with restriction sites.
Materials: Materials were the same as those in 2.1-D.
Procedure: The procedure is basically the same as 2.1-D.
E: Transformation into E.coli Competent Cells
Goal: Expression of plasmid in Escherichia coli.
Materials: Materials were the same as those in 2.1-E.
Procedure: The procedure is basically the same as 2.1-E.
F: Colony PCR
Goal: Verification whether the strain is constructed correctly.
Materials: Materials were the same as those in 2.1-F.
Procedure: The procedure is basically the same as 2.1-F.
G: Plasmid Extraction
Goal: Obtain the successfully constructed plasmid.
Materials: Materials were the same as those in 2.1-G.
Procedure: The procedure is basically the same as 2.1-G.
H: Transforming plasmid into Pichia pastoris.
Goal: Make lysozyme protein express in Pichia pastoris.
Materials: Materials were the same as those in 2.1-H.
Procedure: The procedure is basically the same as 2.1-H.
⦁ Construction of pPICZα A/ pPICZα A/Buforin II
A: Obtain the target gene
Goal: Obtaining a gene fragment for constructing antibacterial peptide Buforin II.
Materials: The specific primers of antimicrobial peptide A were designed,
and the other materials were the same as those in 2.1-A.
Procedure: The procedure is basically the same as 2.1-A, and the
PCR extension time is adjusted according to the fragment length.
B: Agarose Gel Electrophoresis
Goal: Verify whether the target gene is constructed correctly
Materials: Materials were the same as those in 2.1-B.
Procedure: The procedure is basically the same as 2.1-B.
C: DNA Gel Extraction
Goal: Obtaining DNA fragments with correct construction.
Materials: Materials were the same as those in 2.1-C.
Procedure: The procedure is basically the same as 2.1-C.
D: Restriction enzyme digestion and ligation
Goal: Obtaining DNA fragments with restriction sites.
Materials: Materials were the same as those in 2.1-D.
Procedure: The procedure is basically the same as 2.1-D.
E: Transformation into E.coli Competent Cells
Goal: Expression of plasmid in Escherichia coli.
Materials: Materials were the same as those in 2.1-E.
Procedure: The procedure is basically the same as 2.1-E.
F: Colony PCR
Goal: Verification whether the strain is constructed correctly.
Materials: Materials were the same as those in 2.1-F.
Procedure: The procedure is basically the same as 2.1-F.
G: Plasmid Extraction
Goal: Obtain the successfully constructed plasmid.
Materials: Materials were the same as those in 2.1-G.
Procedure: The procedure is basically the same as 2.1-G.
H: Transforming plasmid into Pichia pastoris.
Goal: Make lysozyme protein express in Pichia pastoris.
Materials: Materials were the same as those in 2.1-H.
Procedure: The procedure is basically the same as 2.1-H.
⦁ Construction of pPICZα A/LL-30
A: Obtain the target gene
Goal: Obtaining a gene fragment for constructing antibacterial peptide LL-30.
Materials: The specific primers of antimicrobial peptide A were designed,
and the other materials were the same as those in 2.1-A.
Procedure: The procedure is basically the same as 2.1-A, and the
PCR extension time is adjusted according to the fragment length.
B: Agarose Gel Electrophoresis
Goal: Verify whether the target gene is constructed correctly
Materials: Materials were the same as those in 2.1-B.
Procedure: The procedure is basically the same as 2.1-B.
C: DNA Gel Extraction
Goal: Obtaining DNA fragments with correct construction.
Materials: Materials were the same as those in 2.1-C.
Procedure: The procedure is basically the same as 2.1-C.
D: Restriction enzyme digestion and ligation
Goal: Obtaining DNA fragments with restriction sites.
Materials: Materials were the same as those in 2.1-D.
Procedure: The procedure is basically the same as 2.1-D.
E: Transformation into E.coli Competent Cells
Goal: Expression of plasmid in Escherichia coli.
Materials: Materials were the same as those in 2.1-E.
Procedure: The procedure is basically the same as 2.1-E.
F: Colony PCR
Goal: Verification whether the strain is constructed correctly.
Materials: Materials were the same as those in 2.1-F.
Procedure: The procedure is basically the same as 2.1-F.
G: Plasmid Extraction
Goal: Obtain the successfully constructed plasmid.
Materials: Materials were the same as those in 2.1-G.
Procedure: The procedure is basically the same as 2.1-G.
H: Transforming plasmid into Pichia pastoris.
Goal: Make lysozyme protein express in Pichia pastoris.
Materials: Materials were the same as those in 2.1-H.
Procedure: The procedure is basically the same as 2.1-H.
⦁ Construction of pPICZα A/Plectasin
A: Obtain the target gene
Goal: Obtaining a gene fragment for constructing antibacterial peptide Plectasin.
Materials: The specific primers of antimicrobial peptide A were designed,
and the other materials were the same as those in 2.1-A.
Procedure: The procedure is basically the same as 2.1-A, and the
PCR extension time is adjusted according to the fragment length.
B: Agarose Gel Electrophoresis
Goal: Verify whether the target gene is constructed correctly
Materials: Materials were the same as those in 2.1-B.
Procedure: The procedure is basically the same as 2.1-B.
C: DNA Gel Extraction
Goal: Obtaining DNA fragments with correct construction.
Materials: Materials were the same as those in 2.1-C.
Procedure: The procedure is basically the same as 2.1-C.
D: Restriction enzyme digestion and ligation
Goal: Obtaining DNA fragments with restriction sites.
Materials: Materials were the same as those in 2.1-D.
Procedure: The procedure is basically the same as 2.1-D.
E: Transformation into E.coli Competent Cells
Goal: Expression of plasmid in Escherichia coli.
Materials: Materials were the same as those in 2.1-E.
Procedure: The procedure is basically the same as 2.1-E.
F: Colony PCR
Goal: Verification whether the strain is constructed correctly.
Materials: Materials were the same as those in 2.1-F.
Procedure: The procedure is basically the same as 2.1-F.
G: Plasmid Extraction
Goal: Obtain the successfully constructed plasmid.
Materials: Materials were the same as those in 2.1-G.
Procedure: The procedure is basically the same as 2.1-G.
H: Transforming plasmid into Pichia pastoris.
Goal: Make lysozyme protein express in Pichia pastoris.
Materials: Materials were the same as those in 2.1-H.
Procedure: The procedure is basically the same as 2.1-H.
⦁ Construction of pPICZα A/Hepcidin
A: Obtain the target gene
Goal: Obtaining a gene fragment for constructing antibacterial peptide Hepcidin.
Materials: The specific primers of antimicrobial peptide A were designed,
and the other materials were the same as those in 2.1-A.
Procedure: The procedure is basically the same as 2.1-A, and the
PCR extension time is adjusted according to the fragment length.
B: Agarose Gel Electrophoresis
Goal: Verify whether the target gene is constructed correctly
Materials: Materials were the same as those in 2.1-B.
Procedure: The procedure is basically the same as 2.1-B.
C: DNA Gel Extraction
Goal: Obtaining DNA fragments with correct construction.
Materials: Materials were the same as those in 2.1-C.
Procedure: The procedure is basically the same as 2.1-C.
D: Restriction enzyme digestion and ligation
Goal: Obtaining DNA fragments with restriction sites.
Materials: Materials were the same as those in 2.1-D.
Procedure: The procedure is basically the same as 2.1-D.
E: Transformation into E.coli Competent Cells
Goal: Expression of plasmid in Escherichia coli.
Materials: Materials were the same as those in 2.1-E.
Procedure: The procedure is basically the same as 2.1-E.
F: Colony PCR
Goal: Verification whether the strain is constructed correctly.
Materials: Materials were the same as those in 2.1-F.
Procedure: The procedure is basically the same as 2.1-F.
G: Plasmid Extraction
Goal: Obtain the successfully constructed plasmid.
Materials: Materials were the same as those in 2.1-G.
Procedure: The procedure is basically the same as 2.1-G.
H: Transforming plasmid into Pichia pastoris.
Goal: Make lysozyme protein express in Pichia pastoris.
Materials: Materials were the same as those in 2.1-H.
Procedure: The procedure is basically the same as 2.1-H.
⦁ Construction of pPICZα A/Piscidin-1
A: Obtain the target gene
Goal: Obtaining a gene fragment for constructing antibacterial peptide Piscidin-1.
Materials: The specific primers of antimicrobial peptide A were designed,
and the other materials were the same as those in 2.1-A.
Procedure: The procedure is basically the same as 2.1-A, and the
PCR extension time is adjusted according to the fragment length.
B: Agarose Gel Electrophoresis
Goal: Verify whether the target gene is constructed correctly
Materials: Materials were the same as those in 2.1-B.
Procedure: The procedure is basically the same as 2.1-B.
C: DNA Gel Extraction
Goal: Obtaining DNA fragments with correct construction.
Materials: Materials were the same as those in 2.1-C.
Procedure: The procedure is basically the same as 2.1-C.
D: Restriction enzyme digestion and ligation
Goal: Obtaining DNA fragments with restriction sites.
Materials: Materials were the same as those in 2.1-D.
Procedure: The procedure is basically the same as 2.1-D.
E: Transformation into E.coli Competent Cells
Goal: Expression of plasmid in Escherichia coli.
Materials: Materials were the same as those in 2.1-E.
Procedure: The procedure is basically the same as 2.1-E.
F: Colony PCR
Goal: Verification whether the strain is constructed correctly.
Materials: Materials were the same as those in 2.1-F.
Procedure: The procedure is basically the same as 2.1-F.
G: Plasmid Extraction
Goal: Obtain the successfully constructed plasmid.
Materials: Materials were the same as those in 2.1-G.
Procedure: The procedure is basically the same as 2.1-G.
H: Transforming plasmid into Pichia pastoris.
Goal: Make lysozyme protein express in Pichia pastoris.
Materials: Materials were the same as those in 2.1-H.
Procedure: The procedure is basically the same as 2.1-H.
⦁ Construction of pPICZα A/Lactoferricin
A: Obtain the target gene
Goal: Obtaining a gene fragment for constructing antibacterial peptide Lactoferricin.
Materials: The specific primers of antimicrobial peptide A were designed,
and the other materials were the same as those in 2.1-A.
Procedure: The procedure is basically the same as 2.1-A, and the
PCR extension time is adjusted according to the fragment length.
B: Agarose Gel Electrophoresis
Goal: Verify whether the target gene is constructed correctly
Materials: Materials were the same as those in 2.1-B.
Procedure: The procedure is basically the same as 2.1-B.
C: DNA Gel Extraction
Goal: Obtaining DNA fragments with correct construction.
Materials: Materials were the same as those in 2.1-C.
Procedure: The procedure is basically the same as 2.1-C.
D: Restriction enzyme digestion and ligation
Goal: Obtaining DNA fragments with restriction sites.
Materials: Materials were the same as those in 2.1-D.
Procedure: The procedure is basically the same as 2.1-D.
E: Transformation into E.coli Competent Cells
Goal: Expression of plasmid in Escherichia coli.
Materials: Materials were the same as those in 2.1-E.
Procedure: The procedure is basically the same as 2.1-E.
F: Colony PCR
Goal: Verification whether the strain is constructed correctly.
Materials: Materials were the same as those in 2.1-F.
Procedure: The procedure is basically the same as 2.1-F.
G: Plasmid Extraction
Goal: Obtain the successfully constructed plasmid.
Materials: Materials were the same as those in 2.1-G.
Procedure: The procedure is basically the same as 2.1-G.
H: Transforming plasmid into Pichia pastoris.
Goal: Make lysozyme protein express in Pichia pastoris.
Materials: Materials were the same as those in 2.1-H.
Procedure: The procedure is basically the same as 2.1-H.
⦁ Construction of pPICZα A/antisepsis mix
A: Obtain the target gene
Goal: Obtaining a gene fragment for constructing antibacterial peptide antisepsis mix.
Materials: The specific primers of antimicrobial peptide A were designed,
and the other materials were the same as those in 2.1-A.
Procedure: The procedure is basically the same as 2.1-A, and the
PCR extension time is adjusted according to the fragment length.
B: Agarose Gel Electrophoresis
Goal: Verify whether the target gene is constructed correctly
Materials: Materials were the same as those in 2.1-B.
Procedure: The procedure is basically the same as 2.1-B.
C: DNA Gel Extraction
Goal: Obtaining DNA fragments with correct construction.
Materials: Materials were the same as those in 2.1-C.
Procedure: The procedure is basically the same as 2.1-C.
D: Restriction enzyme digestion and ligation
Goal: Obtaining DNA fragments with restriction sites.
Materials: Materials were the same as those in 2.1-D.
Procedure: The procedure is basically the same as 2.1-D.
E: Transformation into E.coli Competent Cells
Goal: Expression of plasmid in Escherichia coli.
Materials: Materials were the same as those in 2.1-E.
Procedure: The procedure is basically the same as 2.1-E.
F: Colony PCR
Goal: Verification whether the strain is constructed correctly.
Materials: Materials were the same as those in 2.1-F.
Procedure: The procedure is basically the same as 2.1-F.
G: Plasmid Extraction
Goal: Obtain the successfully constructed plasmid.
Materials: Materials were the same as those in 2.1-G.
Procedure: The procedure is basically the same as 2.1-G.
H: Transforming plasmid into Pichia pastoris.
Goal: Make lysozyme protein express in Pichia pastoris.
Materials: Materials were the same as those in 2.1-H.
Procedure: The procedure is basically the same as 2.1-H.
Functional Test
⦁ The growth curve of Pichia pastoris
Goal: The growth curve measurement was used to verify whether the growth ability
of yeast was different when different genes were introduced.
Materials:
LB liquid medium
Pichia pastoris
Bleomycin Zecoin
Procedure:
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..
⦁ SDS-PAGE of antimicrobial peptide protein and lysozyme protein
Goal: Antimicrobial peptides and lysozyme proteins were obtained by protein
induction and verified by SDS-PAGE.
Materials:
gloves
lab coat
parallel pipette
pipette tip
dd water
electrophoresis apparatus
30% Acr-Bis
Lower gel buffer
Upper gel buffer
10% Gel polymerization catalyst
TEMED Substitute
Tris
SDS
Glycine
His-tag Purification Resin
Procedure:
⦁ Inoculate the strain into 3 mL of YPD liquid medium and add bleomycin Zecoin, then incubate at 30°C for 24 h to obtain the primary seed culture.
⦁ Inoculate the primary seed culture into 100 mL of BMGY medium at a 2% inoculum rate, incubate at 30°C until the OD600 reaches approximately 1.5, and measure the growth curve.
⦁ Then centrifuge at 4°C and 5000 rpm for 5 minutes, remove the supernatant, and collect the cells. Resuspend in 50 mL BMMY medium and incubate at 30°C, adding 10 g/L methanol every 24 hours during the incubation period.
⦁ Harvest the cells after 72 hours of protein induction.
All following experiment below was conducted with an ice bath
⦁ Collect the previous bacterial colonies, centrifuge at 6000 rpm for 3 minutes
⦁ Collect the supernatant liquid, then store under -20°C
Strain lysis:
⦁ Combine 4 μL of lysis buffer, lysozyme solution(1mg/ml) under 0°C, let it sit for 30min
⦁ Supersonic lysis under 300W power for 5min (the sonication process consists of a cycle, shifting between 5-second sonication and 5-second breaks).
⦁ Centrifuge at 6000 rpm under 4°C Celsius for ten minutes.
⦁ The combination of 4ml supernatant liquid and 1ml of Purification Resin (BeyoGoldTMHis-Tag), let it sit for 2 hours.
⦁ Transfer the formulae into an affinity layer adsorption column, collect the filtered liquid, and label the substance as liquid number 1.
⦁ Wash the affinity column again four times using the wash buffer and collect the liquid in four separate EP tubes.
⦁ Then, wash the same column four more times using the elution buffer, and collect the filtered liquid in four other EP tubes.
⦁ To evaluate the resulting proteins, we ran SDS-Page.
Make SDS-Page gel
Separation gel:
Distilled Water: 4.8 mL(To adjust volume and provide a solvent environment)
30% Acr - Bis (29:1): 10.0 mL(Acrylamide - Bisacrylamide mixture, main component for gel polymerization)
Lower Gel Buffer (4X): 5.0 mL(Provide appropriate pH and ionic strength for separating gel)
10% Gel Polymerization Catalyst:0.2 mL(Initiate gel polymerization reaction, usually contains ammonium persulfate)
- TEMED Substitute: 0.008 mL(Accelerate the polymerization process, analogous to TEMED function)
Staking gel:
- Distilled Water:2.33 mL(Solvent and volume adjustment)
- 30% Acr - Bis (29:1):0.67 mL(Acrylamide - Bisacrylamide for stacking gel structure)
- Upper Gel Buffer (4X): 1 mL(Provide suitable pH and ionic strength for stacking gel)
- 10% Gel Polymerization Catalyst:0.04 mL(Trigger stacking gel polymerization)
- TEMED Substitute:0.004 mL(Speed up stacking gel formation)
Electrophoresis Solution:Tris 3g, Glycine 14.4g, and SDS 1g. Add double-distilled water to bring the volume up to 1L. When using, dilute it 5 times to obtain the 1×working solution.
Run electrophoresis: run at 80V for 20 minutes, then switch to 180V and run for an additional 40 minutes.
⦁ In order to observe the proteins visually, we dyed them using CBBS (Coomassie brilliant blue staining).
⦁ Determination of Minimum Inhibitory Concentrations of antimicrobial peptides and lysozyme
Goal: Verify the bactericidal activity of antimicrobial peptides and lysozyme.
Materials:
Escherichia coli K-12
Bacillus subtilis
LB culture medium
Petri dishes
Microcentrifuge tubes
Pipette tips
Sterile water
Incubator
Ampicillin
microplate reader
96-well bacterial culture plate
Procedure:
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.
⦁ Time-Kill curve of antimicrobial peptides and lysozyme
Goal: Verify the bactericidal activity of antimicrobial peptides and lysozyme.
Materials:
Bacillus subtilis
Escherichia coli
Lab coat
Gloves
EP tube
Lysozymes
Antibacterial peptides
Pipette and tips
Cell culture plate
Procedure:
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.
⦁ Laser Scanning Confocal Microscope observation
Goal: The bactericidal effect of antimicrobial peptides and lysozyme was
determined by living dead cell staining.
Materials:
Escherichia coli K-12
Bacillus subtilis
LB culture medium
Petri dishes
Microcentrifuge tubes
Pipette tips
Sterile water
Incubator
Ampicillin
Procedure:
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.
⦁ Morphological observation of scanning electron microscope
Goal: The morphology of bacteria treated with antimicrobial peptides and lysozyme was observed by scanning electron microscope.
Materials:
Escherichia coli K-12
Bacillus subtilis
LB culture medium
Petri dishes
Microcentrifuge tubes
Pipette tips
Sterile water
Incubator
Ampicillin
Procedure:
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.