| Medium | Materials | Mass/g |
|---|---|---|
| LB Medium (1000mL) | Tryptone | 10 |
| NaCl | 10 | |
| Yeast Extract | 5 | |
| LB Agar (1000mL) | Tryptone | 10 |
| NaCl | 10 | |
| Yeast Extract | 5 | |
| Agar | 15 | |
| HS Medium (500mL) | Tryptone | 2.5 |
| Yeast Extract | 2.5 | |
| Glucose | 10 | |
| H25Na2O16P | 3.4 | |
| C6H10O8 | 0.75 | |
| HS Agar (500mL) | Tryptone | 2.5 |
| Yeast Extract | 2.5 | |
| Glucose | 10 | |
| H25Na2O16P | 3.4 | |
| C6H10O8 | 0.75 | |
| Agar | 7.5 |
1. Set antibiotics until room temperature from -20℃ refrigerator, label the centrifuge tubes, microfiltration membranes and syringes, calculate and record the mass of the antibiotics; * Note: Calculation method: Preparation Volume (mL) * Concentration (mg/mL) = Mass (mg) 2. When antibiotics thaw to room temperature, start weighing the antibiotics with analytical balance and dissolve it with pure water; 3. After dissolving, filter the solution through 0.22μm millipore filter and store it at 4℃. * Note: Chloramphenicol should be dissolved in absolute ethanol.
1. Weigh 1g Cellulose powder into a 50mL centrifuge tube and add 20mL ddH2O to dissolve it. Ultrasonic can accelerate dissolution. After dissolution, there will be a lot of foam in the centrifuge tube, which affects our final volume calibration. 2. Centrifuge at 8000rpm for 10min. The centrifugal time depends on the foam volume so that it does not affect volume calibration. You can take out the centrifuge tube to observe every 10min. 3. After there is not much foam, calibrate the volume to 25mL and then pass through the membrane.
1. Add 25μL 2 × Phanta Max Buffer, 18μL ddH2O, 1μL dNTP Mix (10 mM each), 1μL Phanta Max Super-Fidelity DNA Polymerase, 1μL DNA templates (need to be diluted to 30ng/μL) 1μL upper primer and 1μL downstream primer, mix and put them into the PCR thermal cycler (ProFlex 3x32-Well). 2. The amplified products were verified by 1% agarose gel electrophoresis, compared with bp and cut their correct range. Then carry out gel recovery: 2.1. Add 300μL Binding Buffer, centrifuge at 12000rpm for 1min, discard waste liquid——repeat twice; 2.2. Add 700μL SPW Wash Buffer, centrifuge at 12000rpm for 1min, discard waste liquid——repeat twice; 2.3. Centrifuge at 12000rpm for 2min; 2.4. Add 30μL ddH2O and stand for 2min, centrifuge at 12000rpm for 1min; 2.5. Measure the DNA concentration. 3. Ligation: Add 2μL 5×CE Multis Buffer, 1μL enzyme (Exnase Multis), appropriate volume of fragments, vectors and ddH2O. And put them into the PCR thermal cycler. * Calculation: (1) The volume of fragments and vectors=(0.02×bp)/Concentration of PCR; (2) The volume of ddH2O=10μL-the volumes of all solutions expected ddH2O. 4. Transformation: 4.1. Thaw the competent cells (on ice), add the ligated products and apply ice for 30min. Heat it for 90sec at 42℃ in a water bath, then put in a ice bath again for 2min; 4.2. Add 700μL LB medium to bacterium liquid, shake it at 37℃ and 220rpm for 1h in a shaker, then centrifuge at 10000rpm for 1min; 4.3. Discard 700μL supernatant solution, resuspend the bacteria with pipetting gun, take 100μL to the plate (LB agar and antibiotic), evenly spread with a cell spreader and culture it overnight at 37℃; 5. The PCR verification: 5.1. Bacterial colonies were picked, to lysed in a water bath at 95℃ for 10min and centrifuge at 5000rpm for 30sec; 5.2. Take out 1μL supernatant and add 5μL buffer, 2μL ddH2O,1μL dNTP, 1μL upper primer and 1μL downstream primer, mix and put it into the PCR thermal cycler. 5.3. The amplified products were verified by 1% agarose gel electrophoresis to compare bp and record the corresponding bacteria. Then select and inoculate bacteria into medium (antibiotic included) and shake them at 37℃ and 220rpm for 12-16h in a shaker. 6. Plasmid extraction: 6.1. Take 1.5mL bacterial solution and centrifuge at 12000rpm for 1min, discard the waste solution ——repeat twice; 6.2. Add 250μL Solution I and resuspend; add 250μL Solution II and shake gently; add 250μL Solution III and shake rapidly, then centrifuge at 12000rpm for 10min; 6.3. Take the supernatant into the absorbent tube, and centrifuge at 12000rpm for 1min, and discard the waste solution; 6.4. Add 500μL HBC Buffer and centrifuge at 12000rpm for 1min, and discard the waste solution; 6.5. Add 700μL DNA Wash Buffer and centrifuge at 12000rpm for 1min, discard the waste solution——repeat twice; 6.6. Centrifuged at 12000rpm for 2min; 6.7. Add 35μL ddH2O, stand for 2min and centrifuge at 12000rpm for 1min; 6.8. Measure the concentration.
6.1. Inoculate Gluconacetobacter hansenii ATCC53582 in 50mL centrifuge tube with 5mL HS medium and 0.2%(v/v) Cellulose, and shake it at 30℃ and 220rpm for 24-72h until OD600>0.7.
6.2. Take 1mL bacterial solution and inoculate to 10mL HS medium and 0.2% Cellulose in a 250mL conical flask, shake it at 220rpm and 30℃ for overnight until OD600=0.4-0.7.
6.3. Pre-cool centrifuge to 4℃, prepare ice bucket and ice, and place 1mM HEPES buffer and 15% glycerol on the ice.
6.4. Bacteria that reached OD600 =0.4-0.7 are put on ice for 10min.
6.5. Centrifuge at 4100rpm and 4℃ for 5min, and discard the supernatant solution.
* Note: Pour out the supernatant solution carefully, but not the pellet. Gluconacetobacter hansenii ATCC53582 doesn’t form precipitates as readily as Escherichia coli (E. coli), most likely due to the buffering effect of Cellulose. If the pellet doesn’t adhere to the tube wall after centrifugation, apply the pellet to the tube wall and centrifuge again for a longer period of time.
6.6. Resuspend with 10mL HEPES buffer.
6.7. Centrifuge at 4100rpm and 4℃ for 5min, discard the supernatant solution. Repeat steps 6.5. to 6.7.
6.8. After resuspending with 5mL of 15% glycerol, place the bacterial solution in a centrifuge at 4℃ for 5min at 4100rpm. Discard the supernatant solution.
6.9. After resuspending the bacteria with 1mL of 15% pre-cooled glycerol, pack each 100μL of it individually into 1.5mL centrifuge tubes.
* Note: After freezing, the transform efficiency of the competent cell may decrease, so immediately use may yield the highest efficiency.
6.10. Add 600ng plasmid to competent cells and flick gently, then transfer the bacterial solution into a (0.1cm) electroporation cup and set 3000V for 5-8ms. After completion, immediately take the bacteria to 800mL HS medium with 0.2%(v/v) Cellulose, and culture it at 220rpm and 30℃ for 16h.
Calculation: the volume of plasmid=600ng/Concentration (no more than 10μL.)
6.11. After centrifuging for 10min, discard 700μL surpernatant solution resuspend with 100μL of HS medium and spread the bacterial solution on the plate with the HS agar (including antibiotic), the colonies will grow for 24-72h. It needs to be verified by PCR to ensure the transform results.
1.Preparation of the kit 1.1. Preparation of GSSG stock solution: 816μL pure water is added to 5mg GSSG provided in this kit, dissolve well and mix well, that is the GSSG stock solution with a concentration of 10mM. Except for the part to be used immediately, the rest of GSSG stock solution should be properly divided and stored at -20°C 1.2. Preparation of DTNB stock solution: 4.5mg DTNB is added to 1.5mL DMSO provided by this kit, dissolve and mix well, that is DTNB stock solution. Except for the immediately to be used part, the remaining DTNB stock solution should be stored at -20°C after proper aliquoting. 1.3. Preparation of protein removal reagent M solution: Weigh 0.2g protein removal reagent M, add 4mL total glutathione detection buffer, and prepare 4mL of 5% aqueous solution, protein removal reagent M solution must be freshly prepared and limited to be used on the same day. 1.4. Preparation of NADPH stock solution (40mg/mL): Add 100μL pure water to the 4mg NADPH provided in this kit, dissolve and mix well, that is NADPH stock solution. Except for the immediately available part, the rest of the NADPH stock solution should be stored at -70°C after proper divided solution. 1.5. Preparation of 5x diluted glutathione reductase solution: Take 50μL glutathione reductase, add 200μL total glutathione detection buffer, mix well, that is 5x dilution of total glutathione reductase. 1.6. Glutathione detection solution:
| Reagent name | One sample | Ten samples | Twenty samples |
| Five-fold dilution glutathione reductase | 6.6μL | 66μL | 132μL |
| NADPH Stock solution | 6.6μL | 66μL | 132μL |
| Total glutathione detection buffer | 150μL | 1.5mL | 3mL |
1.7. Preparation of 0.5mg/mL NADPH: Take 10μL NADPH stock solution, add 790μL total glutathione detection buffer, and mix well. 1.8. Preparation of diluted GSH removal auxiliary solution: Add 53μL GSH removal auxiliary solution to 47μl grade pure water and mix immediately. 1.9. Preparation of GSH removal reagent working solution: 89.2μL absolute ethanol is added to 10.8μL GSH scavenger reagent and mixed immediately. The GSH scavenger working solution must also be freshly prepared each time. 2. Preparation of samples 2.1. Take 1.5mL of bacterial liquid, centrifuge at 12000rpm for 1min, discard the waste liquid - repeat 3 times; 2.2. PBS washes bacteria collect bacteria by centrifugation at one time discard the supernatant; 2.3. Add three times of the bacteria volume of protein removal reagent M solution and fully vortex; 2.4. With liquid nitrogen and a 37 degree water bath, the samples are subjected to two rapid freeze-thaws, four degree or an ice bath for five minutes. 2.5. Centrifuge at 10,000rpm for 10min at 4°C and take the supernatant for the determination of total glutathione. 3. Preparation of samples with GSSG content to be detected: 3.1. Take the part of the sample prepared above with the total glutamine content to be detected, add 20μl diluted GSH scavenging auxiliary solution in proportion to each 100μM sample, add diluted GSH scavenging auxiliary solution, and immediately vortex; 3.2. Then add 4μL GSH removal reagent working solution per 100μL sample add GSH clearance working solution and mix well immediately. React for 60min at 25℃; 4. Preparation of the standard samples: Dilute the 10mM GSSG stock solution with protein removal reagent M solution to 15μM GSSG solution, anD then dilute it to 10, 5, 2, 1, 0.5 μM GSG solution sequentially, measure the absorbance of 15, 10, 5, 2, 1, 0.5μM solution and plot the standard curve; 5. Determination of GSSG content in standards: 5.1. Dilute the 10mM GSSG stock solution with protein removal reagent M solution into 15μM GSSG solution, and then dilute it into 10, 5, 2, 1, 0.5μ MGSSG solution sequentially, and take six points of 15, 10, 5, 2, 1, 0.5μM solution as the standard curve. 5.2. Add diluted GSH removal auxiliary solution according to the proportion of adding 20μL diluted GSH removal auxiliary solution per 100μL standard samples and immediately vortex; 5.3. Then add GSH scavenger working solution in the proportion of adding 4μL GSH scavenger working solution per 100μL samples, immediately vortex. React at 25℃ for 60min; 6. Determination of samples and standards samples: 6.1. Refer to the table below, use a 96-well plate, add samples and standards samples in turn, and mix well. Add 150μL total glutathione detection working solution, mix well and incubate at 25°C or room temperature for 5min;
| Reagent name | Blank control | Standard curve | Sample |
| Samples or standards | 0μL | 10μL | xμL |
| Protein removal reagent M | 10μL | 0μL | 10－xμL |
| Total glutathione detection working fluid | 150μL | 150μL | 150μL |
| 0.5mg/mL NADPH | 50μL | 50μL | 50μL |
6.2. Add 50μL 0.5mg/mL NADPH solution and mix well, incubate at 25°C or room temperature for 20min; 6.3. Immediately determine A412 with enzyme calibration.
| Compose | Mass/g |
| 1%Carbomer | 0.4 |
| ddH2O | 1.6 |
| 5 times diluted TEA | 0.01 |
| Bacterial solution | 2 |
| Tween 80 | 0.1 |
[1] Green, M. R. Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, 2014).
