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Double enzyme digestion


Materials

Spe I
EcoR I
pSB1A2
10×Buffer
ddH2O


Protocol

  1. Prepare the reaction solution according to the following table：

    EcoR I
    1 µl

    Spe I
    1 μl

    pSB1A2
    2 µg

    10×Buffer
    5 µl

    ddH2O
    to 50 µl

  2. 37 ℃ water bath insulation for 50 minutes


PCR for inserts


Materials

Template
2×Phanta
Primer F
Primer R
ddH2O


Protocol

  1. Prepare the reaction solution according to the following table：

    Template
    10 ng

    2×Phanta
    25.0 µl

    Primer F
    2.0 µl

    Primer R
    2.0 µl

    ddH2O
    to 50.0 µl

  2. Run the PCR reaction：

    beaker

Recombination


Vazyme | ClonExpress MultiS One Step Cloning Kit（C113）

Materials

Linearized vector
Insert
5 × CE MultiS Buffer
Exnase MultiS
ddH2O


Protocol

  1. Dilute the vector and insert fragments at an appropriate ratio before preparing the recombination reaction system, and the amount of each component is not less than 1 µl.

  2. Prepare the following reaction on ice:

  3. Gently pipette up and down for several times to mix thoroughly (DO NOT VORTEX!). Centrifuge to collect the reaction solution to the bottom of the tube.

  4. Incubate at 37℃ for 30 min and immediately chill the tube at 4℃ or on ice.


Transformation

  1. Thaw the competent cells on ice (e.g., DH5α Competent Cell, Vazyme #C502).

  2. Pipette 10 μl of the recombination products to 100 μl of competent cells, flick the tube wall to mix thoroughly (DO NOT VORTEX!), and then place the tube on ice for 30 min.

  3. Heat shock at 42℃ water bath for 45 sec and then immediately place on ice for 2 - 3 min.

  4. Add 900 μl of SOC or LB liquid medium (without antibiotics). Then, shake at 37℃ for 1 h at 200 - 250 rpm.

  5. Preheat the corresponding resistant LB solid medium plates in a 37℃ incubator.

  6. Centrifuge the culture at 5,000 rpm (2,400 × g) for 5 min, discard 900 μl of supernatant. Then, use the remaining medium to suspend the bacteria and use a sterile bent glass rod to gently spread on the plate which contains the appropriate selection antibiotic.

  7. Incubate at 37℃ for 12 - 16 h.


Colony PCR


Vazyme | 2 × Rapid Taq Master Mix（P222）

Materials

2 × Rapid Taq Master Mix


Protocol

  1. Prepare the reaction system

    ddH2O
    to 50.0 µl

    2 × Rapid Taq Master Mix
    25.0 µl

    Primer 1 (10 µM)
    2.0 µl

    Primer 2 (10 µM)
    2.0 µl

    Template DNA*
    x µl

    Optimal reaction concentration for plasmid DNA is 0.1 - 10 ng.

  2. Run PCR reaction

    a. The initial denaturation conditions are suitable for most amplification reactions and can be adjusted according to the complexity of the template structure. If the template structure is complex, the initial denaturation time can be extended to 5 - 10 min to improve the initial denaturation effect.
    b. The annealing temperature needs to be adjusted according to the Tm value of the primer, generally set to be 3 ~ 5℃ lower than the Tm value of the primer; For complex templates, it is necessary to adjust the annealing temperature and extend the extension time to achieve efficient amplification.
    c. For higher yields, the extension time can be set to 2 - 5 sec for PCR reactions with products less than 1 kb; for PCR reactions with products more than 1 kb, the extension time can be extended to 20 - 30 sec/kb.


Agarose Gel Electrophoresis


Materials

GelRed nucleic acid dyes
DL2000 DNA Marker
DL5000 DNA Marker
PCR products
Double digestion products


Protocol

  1. Add 1.2g of agarose and 80ml of 1×TAE buffer to a Erlenmeyer flask, and swirl to suspend the agarose powder in the buffer.

  2. Place the gel solution into the microwave. Using a low to medium setting, set the timer for a minimum of 5 minutes, stopping the microwave oven every 30 seconds and swirling the flask gently to suspend the undissloved agarose.

  3. Boil and swirl the solution until all of the small translucent agarose particles are dissolved.

  4. Set aside the flask to cool to 60℃ before adding 2μl GelRed nucleic acid dyes.

  5. Place the comb into the 15×7 cm slot of the tray and pour the molten agarose into the gel tray.

  6. Allow 30-40 minutes for the gel to solidify at room temperature.

  7. Carefully remove the comb from the solidified gel and remove the tape from the edges of the gel tray.Place the tray onto the leveled Sub-Cell so that the sample wells are near the cathode.

  8. Submerge the gel beneath 2 to 6 mm of 1× TAE buffer.

  9. Load the samples and the marker.

  10. Close the lid of gel tank and apply a voltage of 75V.

  11. When the DNA samples or dyes have migrated a suffcient distance through the gel, turn off the electric current and remove the leads and lid from the gel tank.

  12. Place the gel on the UV transilluminator for nucleic acid visualization and analysis.


Extract the plasmid


Vazyme | FastPure Plasmid Mini Kit（DC201）

Materials

RNase A
Buffer P1
Buffer P2
Buffer P3
Buffer PW1
Buffer PW2
Elution Buffer
FastPure DNA Mini Columns
Collection Tubes 2 ml

Protocol

  1. Transfer 1 - 5 ml of overnight (12 - 16 h) culture to a centrifuge tube (not provided), and centrifuge at 10,000 rpm (11,500 × g) for 1 min. Discard the culture medium and place the tube inverted on a blotting paper to drain the liquid.

  2. Add 250 μl of Buffer P1 (make sure that RNase A has been added) to the centrifuge tube containing the precipitated bacterial cells, and mix thoroughly by pipetting or vortexing.

  3. Add 250 μl of Buffer P2 to the mixture from Step 2. Mix by gently inverting the tube 8 – 10 times to completely lyse the cells.

  4. Add 350 μl of Buffer P3 to the mixture from Step 3. Immediately invert the tube gently 8 – 10 times to fully neutralize Buffer P2. At this time, white flocculent precipitates should form. Centrifuge at 12,000 rpm (13,400 × g) for 10 min.

  5. Place FastPure DNA Mini Columns into a Collection Tube 2 ml. Carefully transfer the supernatant from Step 4 to the FastPure DNA Mini Columns with a pipette, taking care not to disturb the precipitates. Centrifuge at 12,000 rpm (13,400 × g) for 30 - 60 sec. Discard the filtrate and place the FastPure DNA Mini Columns back into the Collection Tube.

  6. (Optional) Add 500 μl of Buffer PW1 to the FastPure DNA Mini Columns. Centrifuge at 12,000 rpm (13,400 × g) for 30 - 60 sec. Discard the filtrate and place the FastPure DNA Mini Columns back into the Collection Tube.

  7. Add 600 μl of Buffer PW2 (make sure that absolute ethanol has been added) to the FastPure DNA Mini Columns. Centrifuge at 12,000 rpm (13,400 × g) for 30 - 60 sec. Discard the filtrate and place the FastPure DNA Mini Columns back into the Collection Tube.

  8. Repeat Step 7.

  9. Place the FastPure DNA Mini Columns back into the Collection Tube. Centrifuge the empty column at 12,000 rpm (13,400 × g) for 1 min to completely remove the residual wash buffer.

  10. Place the FastPure DNA Mini Columns in a new sterile 1.5 ml centrifuge tube. Add 30 - 00 μl of Elution Buffer to the center of the spin column membrane. Leave the system at room temperature for 2 min, and centrifuge at 12,000 rpm (13,400 × g) for 1 min to elute the DNA.

  11. Discard the FastPure DNA Mini Columns and store the extracted DNA at -20°C to prevent degradation.

Preparation of Competent Komagataeibacter xylinus


Materials

Komagataeibacter xylinus ATCC 700178
sterile pre-chilled water
sterile pre-chilled 10% glycerol

Protocol

  1. Inoculate the bacteria into 5ml of culture medium and cultivate on a shaker at 30°C for 2 days.
    The following steps must be carried out entirely on ice and within a clean bench.

  2. Inoculate the cultured bacteria into 100ml of culture medium at a ratio of 1:100. Cultivate at 30°C with agitation at 180rpm until the optical density (OD) reaches 0.4-0.6.

  3. Place the culture on ice for 30 minutes, then transfer it into 50ml EP tubes within the clean bench.

  4. Centrifuge at 6000rpm at 4°C for 10 minutes and discard the supernatant.

  5. Add 35ml of sterile pre-chilled water to resuspend the bacterial pellet.

  6. Centrifuge again at 6000rpm at 4°C for 10 minutes and discard the supernatant.

  7. Resuspend the bacterial pellet in 20ml of sterile pre-chilled water.

  8. Repeat the previous step.

  9. Suspend the bacterial pellet in 2.5ml of sterile pre-chilled 10% glycerol, and aliquot 150μL into 1.5ml EP tubes. Store at -80°C for later use.
    Calcium ions can be added for optimization during the preparation of competent cells.

Electroporation


Materials

ddH2O
75% ethanol
absolute ethanol
competent cells
recombinant plasmid


Protocol

  1. Clean the 0.2 mm electroporation cuvette with ddH2O, followed by 75% ethanol, and then absolute ethanol.

  2. Thaw a certain amount of competent cells (approximately 40μL) on ice, add 6μL of recombinant plasmid (specific volume to be determined). After a 30-minute ice bath, transfer the mixture into the electroporation cuvette, taking care to avoid bubble formation.

  3. Perform electroporation at 2.5kV for 5ms. In the clean bench, add 1ml of culture medium, then transfer the mixture into a 1.5ml EP tube and incubate at 30°C with shaking at 180rpm for recovery.

  4. After 7 hours, centrifuge at 8000rpm for 1 minute, discard 900μL of the supernatant, resuspend the pellet, and streak onto a selective solid culture medium. Incubate at 37°C.

Preparation of HS culture medium


Materials

Glucose
Peptone
Yeast extract
Na2HPO4
Citric acid
Distilled water


Protocol

  1. Rinse the graduated cylinder and the large beaker three times with distilled water.

  2. Prepare HS culture medium according to the following formula.

    Glucose
    20 g/L

    Peptone
    5 g/L

    Yeast extract
    5 g/L

    Na2HPO4 (Sodium hydrogen phosphate)
    2.7 g/L

    Citric acid
    1.15 g/L

  3. Add water to reach 800-900ml.

  4. Place it on a magnetic stirrer, stir, then transfer it to a graduated cylinder. Add water to reach 1L and pour it back into the beaker.

  5. Briefly sonicate the solution to make it clear.

  6. Distribute into two conical flasks, each with 100ml, and store the remaining 800ml in a glass bottle.

  7. Autoclave at 115°C (including glucose, so it cannot exceed 115°C, as it may caramelize) for 20 minutes.


Measurement of the K. xylinus growth curve


Materials

bacterial culture medium
cellulase


Protocol

  1. Take 3 sterilized 250 ml Erlenmeyer flasks, labeled 1, 2, and 3, and add 50 ml of HS culture medium respectively.

  2. Set the initial OD value of the bacterial solution to 0.02. The volume of bacterial culture medium to be added is calculated.

  3. Add cellulase in the bottle at 1:1000, respectively.

  4. Seal the Erlenmeyer flask, put it into a shaker for culture, and take out 3-4ml of bacterial culture medium every 3 hours to measure the OD value.