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ASTWS-China

Protocols

Welcome to iGEM 2021

1LB Liquid Culture Medium

Materials for 5L:

Yeast Extract: 25 g

Tryptone: 50 g

NaCl: 50 g

 

Steps

1)     Measure 50 g of Tryptone, 25 g of Yeast extract, and 50 g of NaCl. Place them in a 5,000 mL sterilized bottle.

2)     Add 4.5 L of deionized water, shake the bottle until the solids completely dissolve. Then, add more deionized water until the solution has a volume of 5 L.

3)     Place the bottles into the autoclave for sterilization, under 121°C for 20 min.

4)     Let the bottles cool down under room temperature. The solution would preserve under room temperature for a maximum of 7 days.

 

2LB Solid Culture Medium

Materials for 300mL:

Ager5g

LB Liquid Culture Medium300mL

50 mg/mL Ampicillin

30 mg/mL Kanamycin

 

Steps

1)     Prepare the LB liquid medium according to the LB liquid medium formula. Before autoclaving, add 300 mL LB liquid medium into a 500 mL Erlenmeyer flask and add 5 g Agarose at the same time.

2)     After autoclaving, put on gloves and take out the culture medium, shake the container to mix Agarose thoroughly (the temperature of the culture medium is very high at this time, be careful of burns).

3)     When the culture medium is cooled to 5060℃, add 300μL of corresponding antibiotics, shake the container and mix well.

4)     Pave a plate (5 mL medium/60 mm petri dish).

5)     Shelf life: 30 days

3Make competent cell

1)     Take a sterile shaker test tube and add 2 mL of LB (without antibiotics) medium to the ultra-clean workbench.

2)     Take DH5αstrain and BL21(DE3) strain from the ultra-low temperature refrigerator (-80℃) and place them on ice. On a clean bench, insert a hot inoculation loop into the frozen bacteria, then insert it into a test tube containing 2 mL of LB medium, and incubate overnight on a shaker at 37 ℃.

3)     Take 0.5 mL of the above bacterial solution and transfer it to an Erlenmeyer flask containing 50 mL of LB medium, and incubate at 37℃, 200 rpm on a shaker for 2-3h.  Determine OD590 to be 0.375 (<0.4~0.6, cell number <10^8/mL, this is a key parameter!).  Except for centrifugation, the following operations are all performed on the ultra-clean workbench.

4)     Divide the bacterial solution into pre-cooled 50ml sterile centrifuge tubes, place on ice for 10 minutes, and then centrifuge at 5000 rpm for 10 minutes at 4°C.

5)     Invert the EP tube to consume the supernatant, add 30 mL of ice-cold 0.1 mol/L CaCl2 solution, vortex the mixer immediately to mix, and place in ice for 30 min.

6)     Centrifuge at 4℃, 5000rpm for 10min, discard the supernatant, suspend in 30mL ice-cold 0.1mol/L CaCl2 solution, and place in ice for 30 min.

7)     Centrifuge at 4℃, 5000rpm for 10min, discard the supernatant, hang 1ml ice-cold 0.1mol/L CaCl2 (10% glycerol) solution, and fill each tube with 100μL to 1.5mL ultra-clean liquid. Workbench centrifuge tube.  It can be used directly as a transformation experiment, or it can be immediately stored in an ultra-low temperature refrigerator at -80°C.

8)     Spray 70% ethanol on the tabletop contaminated by bacteria, wipe the tabletop dry, and write an experiment report.

 

4Transformation

1)     Adjust the temperature of the constant temperature water bath to 42°C. in advance.

2)     Take out a tube (100μL) of competent bacteria from the -70℃ultra-low temperature freezer, immediately use your fingers to warm and melt it and insert it on the ice, and ice bath for 5-10 min.

3)     Add 5μL of the ligated plasmid mixture (the DNA content does not exceed 100ng),  shake gently, and place on ice for 20 min.

4)      Shake gently and insert it into a 42℃ water bath for 1~2 min for heat shock, then quickly put it back on ice and let it stand for 3~5min.

5)     Add 500μL of LB medium (without antibiotics) to each of the above-mentioned tubes in the ultra-clean workbench and mix gently, then fix it on the spring rack of the shaker, and shake at 37°C. and 120rpm for 1h.

6)     Take 100~300μL of the above conversion mixture in the ultra-clean workbench, drop them into a solid LB plate petri dish containing appropriate antibiotics, and coat them evenly with a glass coating rod burned in an alcohol lamp (Note: glass coating  Wait for a while after the alcohol on the stick goes out, wait for it to cool down before applying).

7)     Mark the painted petri dish and place it in a 37°C. constant temperature incubator for 30-60 minutes until the liquid on the surface penetrates into the medium, then put it upside down and put it in the 37°C. constant temperature incubator overnight  .

8)      Spray 70% ethanol on the tabletop contaminated by bacteria, dry the tabletop, and write an experiment report.

9)     Observe the colony clones that grow on the plate, and it is better that the colonies can be separated from each other.  Note the white plaque.

5Colony 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 ℃ .

6DNA electrophoresis

Materials:

0.3g agarose

30ml 1x TAE

Step

1)     Mix 0.6ml 5x TAE and 29.4ml H2O to make 30ml 1x TAE

2)     Add 0.3g agarose into 30ml 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 5μl sample with 1 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

7Bacteria preservation

1)     Add 100μl of 80% sterile glycerol.

2)     Add 400μl of the cell we want to preserve.

3)     Mix them together to ensure they are mixed evenly.

4)     Write important information like date, parts, bacteria species, the operation stuff and so on.

8Plasmid extraction

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 SDS-PAGE

1prepare

A. 30%W/VAcrylamide29 g Acrylamide, 1 g BIS, add ddH2O to 100mL, 0.45μM filtration to remove impurities, store in 4℃ with a brown bottles.

B. 5Í TGB SDS-PAGE bufferTris15.1 g, Glycine94 g, SDS5 g. add 800mL ddH2O, store in room temperature.

C. 10%W/VAPS1 g ammonium persulfate, add 10mL ddH2O, store in 4℃.

D. 5ÍSDS-PAGE Loading Buffer: 1M Tris-HCl(pH6.8) 1.25 mL, SDS 0.5 g, Bromophenol BlueBPB25 mg, Glycerol 2.5 mL, add ddH2O to 5ml, store in room temperature.

E. Separating Gel: 10 mL of 10% gel, 30% acrylamide, 4mL 4Í Tris-Cl/ SDS: 2.5mL (pH 8.8), 10% APS: 0.1mL, TEMED: 4μL , ddH2O: 3.4mL.

F. Stacking Gel 5mL: 30% acrylamide: 0.83mL, 4Í Tris-Cl/SDS(pH6.8): 0.625mL, 10% APS: 50μL , TEMED: 5μL , 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.

 

10. 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.

 

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. 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 45s.

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.

 

13. HPLC

1) Prepare

Standard STD:The concentration of  MHET is 1/1.5/3/6 μM respectively.

Group 1---MHETase(E. coli)+MHET(the concentration of MHET is 0.25/0.5/1/2/4/8 μM respectively.)

Group 2---OMPR(E. coli)+MHETase(E. coli)+MHET(the concentration of MHET is 0.25/0.5/1/2/4/8 μM respectively.)

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

 

2) HPLC method

Analyte analysis of samples was performed on an Thermo LC system. Each sample and standard were injected using a volume of 20 μL onto C18 column. The column temperature was maintained at 40°C and the mobile phase used to separate the analytes of interest was composed of phosphoric acid in water. Five calibration standards were used with an r2 coefficient of 0.995 or better and a calibration verification standard (CVS) to ensure the integrity of the initial calibration. Samples were diluted with an equal volume of ultrapure water for analysis.

 

14. Western blotting

Total proteins were extracted using the protein extraction reagent (Pierce) supplemented with a protease inhibitor cocktail (Roche Diagnostics) and separated by SDS-PAGE. The resolved proteins were transferred to PVDF membranes (Millipore). Antibodies were used as follows: Anti-HisTag (Oasisbiofarm Inc; 1:1,000).

 

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