Cloning of plasmids was conducted in following procedures using E.coli.
Recombine DNA fragments with SLiCE (Seamless Ligation Cloning Extract) method and culture over a night on a LB Amp agar plate.
Make sure that the plasmid was successfully inserted with colony PCR, and then culture over a night in liquid LB Amp.
Extract plasmids from cultured E. coli.
SLiCE
Component
Amount
Insert DNA (20 ng/μL)
3 μL
Linear vector backbone (20ng/μL)
3 μL
10x Buffer
1 μL
SLiCE
1 μL
Insert DNA (20ng/μL)
3 μL
Water
up to 10 μL (2 μL)
Components listed above are mixed in a 1.5mL tube, then incubated for 30 min at 37°C.
Transformation was conducted using this mixture as a DNA vector.
Transformed E. coli was spread on the LB agar plate.
Transformation
Component
Amount
JM109 (E. coli competent cell)
1 tube
Soc medium
1 mL
DNA vector
Proper quantity
The DNA vector was added to the tube containing JM109 melted on ice and mixed by tapping.
The tube was put on ice for 30 minutes.
The tube was put on a thermal cycler 45 sec at 42°C, then transferred on ice.
After more than two minutes, 1mL of 36°C Soc medium was added to the tube and mixed by pipetting.
The incubation was conducted for about 30 minutes at 37°C.
iVEC
Component
Amount
Insert DNA (HBD3 or CYC1, 100 ng/μL)
1~2 μL
Linear vector backbone (PCR product from pRS316)
1~2 μL
E. coli competent cell
LB medium
1 mL
The vector and the insert DNA were added to the tube containing the competent cell melted on ice and mixed it by pipetting softly.
The tube was put on ice for 20 minutes.
1 mL of LB medium was added to the tube and the tube was inverted to mix it.
The tube was incubated for 45 minutes at 37°C, then centrifuged (5000 g, room temperature, 1 min).
The supernatant was discarded, leaving about only 100 μL.
The solution of the competent cell was suspended, spread on the LBAmp agar, and cultured overnight at 37°C.
PCR
PrimeSTAR
Used for PCR which require accuracy, such as duplication of plasmid backbones.
Component
Amount
PrimeSTAR HS DNA Poly
0.5 μL
5x PrimeSTAR Buffer (Mg2+ Plus)
10 μL
dNTP Mixture
4 μL
Forward Primer (10-15 pm)
1 μL
Reverse Primer(10-15pm)
1μL
Water
up to 50 μL
DNA template (< 200 ng)
1.5 μL
Phase 1
Phase 2
Phase 3
Phase 4
98°C
Cycle setting：98°C→55°C(Tm value-5°C)→72°C
72°C
4°C (15°C)
30 sec
10 sec→5 sec or 15 sec→1 min/kb
5 min
∞
The mixture of the components listed above are put on a thermal cycler and the reaction was started.
After the reaction, 2μL of the products was mixed with loading dye and used for electrophoresis to check the specificity of amplification.
GoTaq PCR
Used for detecting expression which does not require accuracy, such as colony PCR.
Component
Amount
2x GoTaq Green Master Mix
10 µL
Water
10 µL
Forward Primer (10-15 pm)
0.5 µL
Reverse Primer (10-15 pm)
0.5 µL
DNA template (liquid or colony)
0.5 µL or one pick
Phase 1
Phase 2
Phase 3
Phase 4
95°C
Cycle setting : 95°C→55°C(Tm value -5°C)→72°C
72°C
4°C(15°C)
30 sec
40 sec→30 sec→1 min/kb
5 min
∞
The mixture of the components listed above are put on a thermal cycler and the reaction was started.
After the reaction, 7μL of the products was used for electrophoresis to check the specificity of amplification.
Purification of PCR products from agarose gel
Wizard SV Gel and PCR Clean-Up System (Promega) was used.
After PCR with PrimeSTAR, a tenth of the amount of 10× loading dye was added to the products.
Electrophoresis was conducted for 32μL of the mixture mixed in step 1.
The gel around the band was cut off for DNA extraction.
DNA was extracted from the piece of agar with Promega A9281.
Inoculation
About 2 mL of LB medium and Ampicillin solution were mixed in a sterilized test tube to set ampicillin concentration to 100 mg / L.
A single colony of the bacteria was poked with a toothpick and dissolved to the medium.
Glycerol stock creation
Component
Amount
E. coli suspension
0.7 mL
50 % Glycerol
0.3 mL
0.7 mL of E. coli suspension was added to 0.3 mL of 50 % Glycerol and mixed by pipetting.
The mixture was stored at -20°C.
Plasmid Extraction
PureYield Plasmid Miniprep System (Promega) was used.
The culture solution in which E. coli cultured overnight was suspended.
Purified the plasmid from the E. coli suspension with Promega A1223.
Medium Composition
LB medium (agar)
Amount
Final conc.
LB Powder
12.5 g
2.5 %
(LB agar)
7.5 g
1.5 %
Water
up to 500 mL
(1000μL of Amp was added to the medium to make LB Amp (agar).)
YPD medium
Amount
Final conc.
Bacto Yeast Extract
2.5 g
1 %
Peptone
5.0 g
2 %
Glucose
5.0 g
2 %
Water
up to 250 mL
BMMD medium (+ Met)
Amount
Final conc.
Bacto Yeast Nitrogen Base w/o Amino Acids
1.68 g
0.67 %
Citric Acid
1.73 g
36 mM
Sodium Hydrogen phosphate
4.471 g
126 mM
Glucose
5 g
2 %
(Methionine)
(37.3 mg)
(1 mM)
Water
up to 250 mL
SC-U medium agar
Amount
Final conc.
Dropout Mix
0.5 g
Bacto Yeast Nitrogen Base w/o Amino
1.68 g
0.67 %
Glucose
5 g
2 %
Bacto agar
5 g
2 %
Water
up to 250 mL
Yeast Transformation
Preculture
3 mL of medium was poured into a tube.
One of the host yeast colonies was poked with the tip of the micropipette.
The tip of the micropipette was soaked and shaken in the medium.
The tube was put into the shaker overnight at 30°C.
In the next morning, the new 3 medium was prepared and 300μL of yeast solution was poured into it.
The yeast was cultured for 3 ~ 4 hours.
Transformation
Following procedure is conducted in accordance with Frozen-EZ Yeast TransformationⅡ (Zymo Research T2001).
After preculture, 1 mL of yeast culture solution was put into a 1.5 mL microtube and centrifuged (3000 rpm, 26°C, 1 min) to separate yeast from the liquid. The supernatant was disposed of.
1mL of Frozen-EZ Sol1(washer solution) was added to the tube. Then the tube was vortexed and centrifuged (3000 rpm, 26°C, 1 min) again. The supernatant was discarded.
2 μL of plasmid solution was added to the tube and the solution was mixed by pipetting and vortex.
500 μL of Frozen-EZ Sol3 was added to the tube and the tube was inverted.
The tube was incubated for 45 min at 30°C. During this time, the tube was inverted every 15 minutes.
After incubation, 150 μL of yeast solution was spread on a plate without mixing.
The plate was cultured for 2 ~ 4 days at 30 °C.
Oxygen promotor
Evaluation of the promoter
Transformed yeast (pANB1) was cultured until saturation.
Two 5 mL syringes filled with 300μL of the yeast medium were shaken at 37 °C overnight.
After 4 hours of cultivation, fluorescence of the yeast was observed visually and fluorescence microscopy.
Defensin evaluation
Operation check of Met17 promotor
200μL of transformed yeast (HBD3, pRS316) solutions were added to YPD medium, after overnight cultivation in BMMD and BMMD +Met medium.
Optical density (OD) of the culture medium was measured using a spectrophotometer at appropriate intervals.
Defensin Assay
Transformed yeast (HBD3, pRS316) was cultured overnight in 6mL of YPD medium. E. coli was also cultured overnight.
3mL of yeast solution was used to make cell fiber.
The remainder of the yeast solution was poured into a tube and centrifuged (1500rpm, 1min).
The supernatant obtained in step 1 was filtered, and the yeast was eliminated totally.
1.5 mL of E. coli was double diluted and spread on a LB agar plate.
Dressing was immersed into the filtered yeast supernatant (or YPD medium as a control) and put on the plate prepared in step 3.
The plate was cultured for 24-48 hours.
Assay Plate preparation
300μL of E. coli solution was diluted 10 times with sterilized saline (0.85 % NaCl).
LBAmp agar was heated in a microwave and melted.
The plate was allowed to cool to 50°C.
1mL of the liquid made in step 1 was added to the plate, and the mixture was stirred.
The plate was left gently until solidified.
Halo Assay
About 30μL of liquid to be evaluated were permeated paper disks.
The paper disks were placed on the assay plates at the center.
The yeast on the plates were cultured for 24-48 hours at 37°C.
For all plates, the diameter of the inhibition circle around the paper disk was measured.
Absorption Method (JIS L 1902:)
E. coli was cultured in LBAmp for 3 hours at 200rpm and 37°C, after overnight cultivation.
The cultured E. coli was diluted 20 times with YPD medium.
One of the dressings to be evaluated (about 0.05g of each dressing) was put in a tube and soaked with 120ul of the 20-fold diluted E. coli culture solution.
12mL of LBAmp was added to the tube and mixed with the solution in the tube by vortex.
3mL of the liquid in the tube was collected and used to make a 10x dilution series, then stored at 5°C to count the number of viable bacteria at that time.
2mL of remaining E. coli solution was collected for another experiment.
The remaining E. coli was cultured 18-24 hours at 36°C and subjected to the same treatment as in step 5.
LBAmp agar was heated in a microwave and allowed to cool to 50°C.
About 20mL of LBAmp agar was poured to dishes and mixed with 1mL of the E. coli dilution series (see also Assay Plate preparation).
The plates were cultured for 24-48 hours at 36°C.
The number of colonies on each plate was counted.
Antimicrobial activity value (AAV) was calculated. AAV = {log(control, viable count after incubation) – log(control, viable count before incubation)} - {log(test, viable count after incubation) – log(test, viable count before incubation)}
Cell Fiber
Cell fiber preparation
200μL of transformed yeast solution was added to the proper medium and cultured for 3 hours at 37°C.
1.5wt% of sodium alginate aq and yeast suspension were mixed, and the mixture was inhaled with a syringe fitted with a 0.8mm needle.
The tip of the syringe was put in the 50mM CaCl2 aq, and the syringe was pushed slowly.
