Team:FAFU-CHINA/Protocol

Team:FAFU-CHINA - 2020.igem.org

1.Molecular biology experiment

1.1Construction

1.1.1 Agarose gel electrophoresis

  1. To separate medium size fragments, the agarose gel is used in a concentration of 1% w/v. Weigh 0.25/0.5/1g of agarose powder in an erlenmeyer flask.
  2. Measure 25/50/100 mL of TBE buffer and add it to the erlenmeyer
  3. Put the mix in the microwave until the agarose is completely dissolved.
  4. Add the gel nucleic stain(Fluorescein) by 0.01%.
  5. Let the mix cool down till it can be added to the electrophoresis tray
  6. When agarose gel is solidified, remove the comb and put the gel into the Electrophoresis Cells filled with TBE buffer.
  7. Add 6X purple loading buffer mixed each DNA sample
  8. Load the DNA samples in the remaining wells(1.5μL for detection, all for purification)
  9. Run the gel with the voltage 200V for 15 min
  10. Visualize the DNA fragments using a UV trans illuminator.

1.1.2 Primer design

1.1.3 Polymerase Chain Reaction

1.1.4 DNA purification after gel electrophoresis

  1. use a clean scalpel or blade to cut out the gel containing the target DNA fragment. The gel should be cut out whenever possible. The gel block containing DNA was placed in a pre weighed 1.5ml centrifuge tube and weighed. Write down the weight of the rubber block. Note: the DNA fragments recovered from X are used for cloning, so it is necessary to avoid damage to DNA by UV irradiation. Minimize exposure time under UV
  2. adding 1:1 volume binding fluid (volume: weight) to the rubber block (for example, adding 100 binding liquid into the gel of 100 mg): injection of agarose gel for T concentration of human I2%, adding 2:1 volume binding liquid to the rubber block.
  3. place the gel mixture at 10 min for 50-60 degrees until the rubber block is completely dissolved. Invert the centrifuge tube every few minutes to speed up the dissolution process. Gel must be completely dissolved. Add a short vortex gel mixture before recovery column. Observe the color of the solution. Yellow indicates the best pH for DNA binding. If the color of the solution is orange or purple, the color of the mixture will turn yellow by adding 10μl 3M of sodium acetate solution.
  4. transfer the 800ul dissolved gel solution (from steps 3 and 4) to the GcneJET recovery column. Centrifuge for 1 minute. Pour out the waste liquid and place the column in the recovery header. Note: if the total volume of gel glue is more than 800 μl., it can be added step by step to the recovery column. After each addition, centrifuge 30-60s and discard the waste liquid until all the gel solutions are added. Each recovery column 1 can exceed 1g of agarose gel.
  5. Optional: this step is suitable for the recovered DNA to be directly used for sequencing. 100μl of binding solution was added to the genejet recovery column. Centrifuge for 1 minute, pour out the waste liquid and put the column into the recovery header.
  6. Add 700 μL of rinse solution to the genejet recovery column. Centrifuge for 1 minute, pour out the waste liquid and put the column into the recovery header,
  7. Centrifuge the empty recovery column for another minute to completely remove the residual rinse solution. Note: this step is to avoid residual ethanol in the recovered DNA. If ethanol remains in DNA, it may inhibit the downstream enzymatic reaction.
  8. Add 30 μ l DD aqueous solution to the center of the membrane, put it into a new EP tube, centrifugate for 1 min at 12000 RMP, repeat once, and store in - 20

1.1.5 Infusion

  • Mix gently and centrifuge to the bottom of the tube
  • Incubate at 37 ℃ for 1 h
  • Ice bath for 5 min
  • Store the product at -20 ℃

1.1.6 transfomation

  1. Add DNA (no more than 50 ng in a volume of 10μL or less)and 100 uL E-coli to each tube , mix the contents of the tubes by swirling gently.
  2. Store the tubes on ice for 30 minutes. Heat shock 90s,42Cwater bath 3 min.
  3. lce bath 5min.
  4. Add 700 μL --900μL LB medium to each tube. Incubate the cultures for 60 minutes in a 37℃shaking table
  5. Then the bacteria was collected by centrifuge(3000rpm,5min).
  6. Transfer 200 μL of transformed competent cells onto agar LB medium containing apm.
  7. Invert the plates and incubate at 37C.Transformed colonies should appear in 12~16 h

1.1.7 Isolation of Plasmid DNA

  1. Inoculate a single colony from an LB plate in 100 ml of LB medium containing the appropriate antibiotic. Incubate overnight at 37℃ with shaking (approximately 225 rpm).
  2. Place 1.2 mL of the overnight culture into a micro-centrifuge tube and centrifuge at 12,000 × g for 30 seconds.
  3. Remove the supernatant and re-suspend the pellet in 100 μL Solution I containing 4 mg/mL lysozyme (at room temperature). Vortex cells into suspension, making sure there are no clumps of cells.
  4. After a few seconds at room temperature, lysozyme action on the cells will be visible: the suspended cells turn slightly milky and tend to coat the sides of the tubes when shaken. Place the tubes into an ice bucket and chill 1 minute.
  5. Add 250 μL of Solution II at room temperature and mix by inversion gently . Return tube to the ice bucket for another minute.
  6. Add 350 μL of ice – cold Solution III, mix, and return to the ice bucket for another 3 minutes. A fluffy white precipitate will form.
  7. Centrifuge at 13,000 × g for 10 minutes in a micro-centrifuge.
  8. Transfer the supernatant to into the column , avoiding the white precipitate.
  9. Add 500 μL HBC buffer ). pour off the waste liquid in the tube
  10. centrifuge at 13,000 × g for 1 minutes.
  11. Transfer the column into the new EP tube and add 50 --100 μL EB buffer into the middle part of the membrane. Centrifuge at 13000 × g for 1 min
  12. asorbing the liquid transfer it into the column and repeat the centrifugation
  13. store the product at -20 ℃

Detect the result by agarose gel electrophoresis

1.1.8 Culture medium preparation

  1. Add 250 ml of water to a 500 ml beaker to volume and mark the liquid line;
  2. Wash the beaker, pour about 200ml of deionized water, and place it on a magnetic stirrer for stirring;
  3. Weigh the medicine into the beaker according to the following ratio
  4. After all the ingredients are completely dissolved, stop stirring and make up the deionized water to the marking line;
  5. Weigh 4.5g agar in a 500ml Erlenmeyer flask at a ratio of 1.8%, and add the prepared medium;
  6. Gauze kraft paper coating, autoclaved at 121 ℃. If the solid LB media is needed, just add 1% w/v of agar into the liquid before autoclaving.

1.1.8 1.1.10 Bacteria liquid PCR

1.2Characterization

1.2.1 Fluorescence detection

1.2.2 Determination of hemicellulose in JUNCAO

1.2.2.1 Preparation of standard solution

The standard was 10 mg D-xylose. Before use, add 1ml distilled water to dissolve and prepare 10mg / ml standard solution.

Dilute 10 mg / ml standard solution with distilled water to 1, 0.9, 0.8, 0.6, 0.4, 0.2 mg / ml standard solution. The preparation method is as follows:

1.2.2.2The hemicellulose content detection kit purchased by Solarbio company was used in the experiment

Calculation of hemicellulose content:

  1. Drawing of standard curve: Taking the concentration of each standard solution as the x-axis and the corresponding Δ a standard as the y-axis, the standard curve was drawn, and the standard equation y = KX + B was obtained. By introducing Δ a into the equation, X (mg / ml) was obtained.
  2. The calculation of the content of half fiber rope is as follows Hemicellulose content (mg / g dry weight) = x × V total + W × f = x + W × f V sample total: volume of added extract, 1ml; W: sample mass, G; F: dilution multiple.

2.Microbiological experiment

2.1.1 Selecting cellulose decomposing bacteria from soil

2.1.1.1 Preparation of CMC solid medium

Each 1000ml medium contained 1g sodium nitrate, 1.5g peptone, 1g potassium dihydrogen phosphate, 0.5g potassium chloride, 0.01g magnesium sulfate heptahydrate, 0.01g ferrous sulfate heptahydrate, 20g sodium carboxymethylcellulose and 20g agar and then sterilized at 121 ℃ and 0.12 MPa

2.1.1.2 Select soil sample

Select soil from areas with high deciduous content

2.1.1.3 Screening colonies

  1. The soil was mixed with 100ml sterile water to obtain 100ml suspension of soil particles.
  2. The supernatant was diluted to 10-2, 10-3, 10-4 and 10-5 times.
  3. 10 μ l of 10-3, 10-4 and 10-5 times of bacterial liquid were absorbed and coated on CMC plate. 4. The plate was placed in 25 ℃ incubator for inverted culture

2.1.2 Congo Red Binding assay

  1. Prepared 0.2% Congo red solution (1000ml)
  2. The selected single colony culture medium was stained
  3. Standing for 30 minutes
  4. The excess dye on the surface of the medium was washed with ddH2O
  5. Observe whether there is a transparent circle around the colony
  6. The single colonies with obvious transparent circle were screened, and the replication plates were preserved for further culture.

2.1.3 DNS method to measure the concentration of reducing sugar

2.1.3.1 DNS

  1. 1 ml bacterial solution was put into EP tube, centrifuged at 8000 R / min for 5 min, and the supernatant was used as crude enzyme solution;
  2. In 150 μ L 1% cellulose solution (dissolved into 0.1 mol / L sodium citrate buffer solution (pH 4.6), 50 μ l supernatant was added as the experimental group;
  3. The supernatant of 50 μ l boiling water bath was added into 150 μ L 1% cellulose solution for 30 min as the control group; the experimental group and the control group were placed in the water bath for 30 min;
  4. Add 150 μ l DNS and 100 μ L NaOH solution into boiling water bath for 5 min; fix the volume to 1 ml, adjust the blank group to zero, measure the od540 value, and take the average value.

2.1.3.2 Preparation of glucose standard curve

Prepare six colorimetric tubes, numbered 1, 2, 3, 4, 5, 6 respectively, add 0, 0.2, 0.4, 0.6, 0.8, 1.0 ml glucose solution of 1 mg / ml, and then add 1.0, 0.8, 0.6, 0.4, 0.2 and 0 ml of ultrapure water, respectively, and add dns3ml, and set two parallel tubes for each tube.

After reaction in boiling water bath for 15 min, it was cooled rapidly and the volume was fixed to 25 ml. under the condition of λ = 540 nm, the absorbance value was measured with a spectrophotometer, and the glucose concentration was taken as the abscissa and the measured absorbance value as the ordinate, and the glucose standard curve was drawn.

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