Team:ASTWS-China/Protocols

Protocols

1、LB Liquid Culture Medium

Materials for 1L:
Yeast Extract: 8 g (Oxoid-LP0021)
Tryptone: 16 g (Oxoid-LP0042)
NaCl: 16 g

Steps:

①　Preparation of LB Liquid Culture Medium
②　Measure 16 g of Tryptone, 8 g of Yeast extract, and 16 g of NaCl. Place them in a 1,000 mL sterilized bottle.
③　Add 0.5 L of deionized water, shake the bottle until the solids completely dissolve. Then, add more deionized water until the solution has a volume of 1 L.
④　Place the bottles into the autoclave for sterilization, under 121°C for 30 minutes.
⑤　Let the bottles cool down under room temperature. The solution would preserve under room temperature for a maximum of 7 days.

2、Preparation of LB Solid Culture Medium

Materials for 1 L:
Yeast Extract: 8 g (Oxoid-LP0021)
Tryptone: 16 g (Oxoid-LP0042)
Agar: 7.5 g
NaCl: 16 g

Steps:

①　Measure 16 g of Tryptone, 8 g of Yeast extract, 16 g of NaCl and 7.5 g of agar . Place them in a 1,000 mL sterilized bottle.
②　 7.5 grams of agar and add the agar into an empty sterilized bottle.
③　Place the agar-containing bottle into the autoclave for sterilization, under 121°C for 10 minutes.
④　Take out the bottle and place it onto the air-clean bench. Let it cool down to approximately 50°C-60°C.
⑤　After cooling down, add 1 mL of 100mg/ml ampicillin to the solution. Add and spread the solution evenly onto culture dishes (20 mL of solution each).
⑥　Seal the culture dishes and preserve them in the freezing counter of the refrigerator.

3、Make competent cell

①　Add 1 mL E.coli DH5α to 100 mL LB liquid medium ,placing the cells on a shaker at 37 °C, 200 rpm for 3 h，OD600nm=0.375.
②　Cool the cells in the ice bath for 10 min.
③　Centrifuge at 5000 rpm at 4 °C for 10 min . Discard the liquid and add 10 ml CaCl2 solution (0.1M).
④　Put the liquid in an ice box and shake it on the shaker for 10 minutes.
⑤　Centrifuge at 5000 rpm for at 4 °C for 10 min. Discard the liquid and add 10 mL of CaCl2 (0.1M) solution.
⑥　Put it in an ice box and shake it on the shaker for 30 minutes.
⑦　Centrifuge at 5000 rpm for at 4 °C for 10 min.
⑧　Transporting each 1.5 mL of the product into the EP tube.

4、Enzyme digestion

Materials:
Plasmid 2µg
Buffer 2µl
EcoRI 0.5µl
BamHI 0.5µl
ddH2O to 20 µl

Steps:

①　Mix all the materials in EP tube
②　Heat the mixture in water bath at 37˚ for 15 min

5、Transformation

①　Thaw 50 μL DH5α/BL21 competent cells on ice (it reach the state of ice water mixture about 5 min which has the best conversion effect)
②　Pipette 3 µL of plasmid into 50 µL thawed cells. (The volume of the transformation should not exceed 1/10 of the volume of the Competent Cells) Gently pipette up and down a 5-6 times. Keep it in ice-bath for 30 min.
③　Heat shock tubes at 42 °C in water bath for 45 s. Incubate on ice for 5 min.
④　Pipette 800 µL LB media to the transformation. Incubate at 37 °C, 150 rpm for 1 h.
⑤　Spin down cells at 3000 rpm for 5 min and discard 600 µL of the supernatant. Resuspend the cells in the remaining 20 µL, and pipette the transformation onto LB plates, spread with sterilized spreader. (please complete the step on the clean bench)
⑥　Incubate transformations overnight at 37 °C.

6、Colony PCR

①　Prepare a few 0.2mL Eppendorf tubes with 20μL ddH2O in each.
②　Choose several single colonies from the agar gel medium to drop in each tube via pipette tips. Stirring the mixture thoroughly to suspend the mixture.
③　Preparing a 20 μL PCR system in PCR tubes: 10 μL PCR Mix Master, 8 μL ddH2O, 0.5 μL suspension ,0.5 μL of each primer mix
④　Protocol of Colony PCR: 95 ℃ preheating for 5 min; Replicating the following steps for 30 times: 95 ℃ heating for 30 s, 50 ℃ annealing for 30 s,72 ℃ extending for 45 s (at a speed of about 1 kbp/min);72 ℃ extending for 3 min; Store the products at 16 ℃ .

7、Bacteria preservation

①　Add 100μl of 80% sterile glycerol.
②　Add 400 μl of the cell we want to preserve.
Mix them together to ensure they are mixed evenly.
Write important information like date, parts, bacteria species, the operation stuff and so on.
Preserve the prepared liquid in -80 ℃.

8、Plasmid extraction

Use OMEGA® E.Z.N.A. Plasmid Mini Kit

①　 Collecting 3 mL bacteria liquid after incubating overnight, centrifuge at 10000 x g at room temperature and discard the medium.
②　Adding 250 μL Solution I (with RNase A), the cells were completely suspended by vortex oscillation.
③　Add 250 μL Solution II to the resuspended mixture and invert it gently for 4-6 times. This reaction should not exceed 5 min.
④　350 μL Solution III was added and reversed several times until white flocculent precipitation was formed.
⑤　10000 × g centrifugation at room temperature for 10 min.
⑥　Transfer the supernatant to a HiBind DNA binding column with 2 mLcollection tube. Centrifuge at 10,000×g for 1 min at room temperature.Drain the filtrate from the pipe.
⑦　Put the column back into the collection tube, add 500 μL HB Buffer, centrifuge at 10,000×g for 1 min at room temperature, discard the filtrate.
⑧　Put the column back into the collection tube, add 700 μL DNA Wash Buffer, centrifuge according to the above conditions, discard the filtrate. Note: The concentrated DNA Wash Buffer must be diluted with anhydrous ethanol prior to use, as indicated on the label
⑨　(optional) to refuse to filtrate, repeat steps 9 once
⑩　Discard the filtrate and reinstall the column into the collection tube. Centrifuge the 10,000 ×g empty column for 2 min to dry the column matrix.
⑪ Mount the column on a clean 1.5 mL centrifuge tube and add 30-50 μL Elution Buffer (10 mM Tris-HCl, PH 8.5) or sterile water to the column matrix. Let it stand for 1-2 min and then Elution out the DNA by centrifugation at 10,000×g for 1 min. The concentration and purity of the plasmid were determined by Nanodrop 2000.

9、DNA electrophoresis

Materials:
0.34g agarose
34ml 1x TAE

Step：

①　Mix 4ml 5x TAE and 196ml H2O to make 200ml 1x TAE
②　Add 0.34g agarose into 34ml 1xTAE
③　Heat the solutions until it becomes transparent
④　Cool the solutions to room temperature and pour them into mould.
⑤　Move the gel plate into electrophoresis tank and soak it into our buffer solutions ( 1XTAE)
⑥　Set baffle and comb in the tank, and seal the baffle inside, until the gel is cooled to about 50˚C
⑦　Remove the baffle and vertically pull out the comb
⑧　Mix each 10 μl sample with 3 μl Gel Red, transfer dye and plasmids to gel sample holes vertically and slowly with pipetting.Immediately start the electrophoresis with a voltage of 120V
⑨　After the electrophoresis, cut off the power and remove the gel. Then observe that is there DNA bands appear neatly under the UV light

10、Gel extraction

Use Gel extraction kit of CWBIO

①　Under ultraviolet light, cut a thin gel piece with needed DNA stripes on it. (try to cut all other extra parts)Then put it in to the 1.5ml centrifuge tube and weigh them together. By subtracting the mass of centrifuge tube, we get the weight of the gel. Then use the function 100mg=100 μl to calculate the volume as 1 gel volume.
②　Add 1 gel volume of Buffer PG
③　Put the solution and the gel into 50℃water and turn the tube upside down for every 2-3 minutes until the solution becomes yellow to ensure the gel is completely dissolved in the solution.
④　Put the Spin Columns DM into the collection tube, add 200μl of Buffer PS and centrifuge at 13000rpm for 1 minute. Then pour all the wasted liquid in the collection tube and put the column into the collection tube.
⑤　Add the solution we get from step 4 to the Spin Columns DM and keep it in the room temperature for 2 minutes. Then centrifuge it at 13000rpm for 1 minute, and pour the wasted liquid and put the column back to the tube.
⑥　Ass 450μl of Buffer PW, centrifuge at 13000rpm for 1 minute and pour the wasted liquid.
⑦　Repeat the step 6
⑧　Centrifuge at 13000 rpm for 1 minute and pour the wasted liquid.
⑨　Put the Spin Columns DM to a new 1.5ml centrifuge tube and add 50μl of Buffer EB above the center of the membrane and keep it in room temperature for 2minutes. Then centrifuge it at 13000 rpm for 1 minute and collect the DNA solution.
⑩　Store it at -20 ℃.

11、p-NP assay

①　Two experimental groups were set up. The first group was inoculated with E. coli expressing only PETase, and the second group was E. coli co-expressing PETsae and OMPR. And then culture at 37°C for 5 hours.
②　Add 7.1 μl of the substrate (4-nitrophenyl butyrate) into 1mL of acetonitrile to prepare a 4mM mother liquor. And then dilute to the concentration of 2/1 / 0.8 / 0.4 / 0.2 / 0.1 mM. 10μL of each was added to a 96-well plate, and another 10μl of acetylene was taken as a blank control.
③　Add 100μl of the bacterial solution to each well and mixing with the substrate. Detect the light absorption value at 405 nm using a microplate reader after mixing for 1min, 2min, 5min, 10min, 15min, 20min.

12、SDS-PAGE

1）prepare:

A. 30%（W/V）Acrylamide：29 g Acrylamide, 1 g BIS, add ddH2O to 100mL, 0.45um filtration to remove impurities, store in 4℃ with a brown bottles.
B. 5X TGB SDS-PAGE buffer：Tris：15.1 g, Glycine：94 g, SDS：5 g. add 800ml ddH2O, store in room temperature.
C. 10%（W/V）APS：1 g ammonium persulfate, add 10ml ddH2O, store in 4℃.
D. 5X SDS-PAGE Loading Buffer: 1M Tris-HCl(pH6.8) 1.25 mL, SDS 0.5 g, Bromophenol Blue（BPB）25 mg, Glycerol 2.5 mL, add ddH2O to 5ml, store in room temperature.
E. Separating Gel: 10 mL of 10% gel, 30% acrylamide, 4mL 4X Tris-CL/ SDS: 2.5mL (pH 8.8), 10% APS: 0.1mL, TEMED: 4uL, ddH2O: 3.4mL.
F. Stacking Gel 5mL: 30% acrylamide: 0.83mL, 4X Tris-Cl/SDS(pH6.8): 0.625mL, 10% APS: 50uL, TEMED: 5uL, ddH2O:3.455mL.

2) Notes:

A. There should be no bubbles in the gel making process. The stacking gel should be poured more than 1ml from the bottom of the comb.
B. The concentration, salinity and pH value of samples should be controlled.
C. Electrophoresis, whether to use ice or refrigerator cooling, depending on the situation.

3) Conditions:

A. Steady current 14 mA.
B. Voltage stabilization: separating gel-120V, stacking Gel-60-80V.

4) Staining:

A. Coomassie brilliant blue dye solution (R250 dye solution): 1g R250, 250mL isopropanol, 100mL glacial acetic acid, 650mL ddH2O, filter paper filter particles, RT storage. The eluent of Coomassie Brilliant Blue: 100mL acetic acid, Ethanol 50mL ddH2O 850mL.
B. Silver staining Gel fixing solution: methanol 500mL, acetic acid 100mL, ddH2O 400mL, RT storage. Gel treatment solution: methanol 50mL, glutaraldehyde 10ml, ddH2O 40mL.
C. Gel dyeing solution: 20% AgNO3 2mL, NH3·H2O 1mL, 4% NaOH 1mL, ddH2O 96mL.
D. Color developing solution: citric acid 50mg, formaldehyde 0.2mL, ddH2O 1 L, RT storage.

13、Congo red stain

1) Culture microorganism, then add Congo red

The colony medium was covered with 1 mg/mL CR solution. After 10-15 min, remove CR solution and add 1 mol/L NaCl Solution. After 15 min, the NaCl solution was poured out. At this time, a transparent circle will appear around the Cellulase Producing colonies.

2) Add Congo red when pouring the plate

Prepare 10 mg/mL CR solution, after sterilization, add 1 mL CR solution to 200 mL medium, mix and pour into the plate. When colonies grow on the medium, a clear circle will appear around the Cellulase Producing colonies.

14、Co-localization of fluorescent protein

1) The 30-100 uL competent cells were mixed with 2-5 uL fluorescent protein particles and placed on ice for 15-30min.
2) 42℃ heat shock for 45-90-120s.
3) Keep on ice for 1-5 minutes.
4) Add 70-800uL LB liquid medium, culture at 37℃ for 30-45-60min.
5) Spread the bacterial solution on the plate.
6) Culture at 37℃ for 12-16 h.
7) Take pictures with a fluorescence microscope, observe and record the expression and co-localization of green fluorescent protein and red fluorescent protein at wavelengths of 488 and 598, respectively.

15、HPLC

1) Sample information

Standard STD: The concentration is 0.001/0.005/0.01/0.05 mM respectively.
Samples: No. 1-stock solution, No. 2-immersion, No. 3-PET+OMP, No. 4-PET

2) HPLC method

Column: zorbax Extend C-18
Injection volume: 20 uL
Column thermostat: 40℃
Flow rate: 1 mL /min
Detection wavelength: 254nm
Mobile phase: 70% MilliQ water, 20% already, and 10% formic acid
Note: It is recommended to run standard No. 4 (0.5mM) first to verify the feasibility of the method and process.

16、Statistics

All experiments were performed in triplicate. Data were analyzed by SPSS12.0 and expressed as means ± SD. Sta- tistical comparisons between two groups were made using an unpaired Student’s t-test and probability values (p) < 0.05 were considered significant.