Team:SZPT-CHINA/Experiments
Experiments
Engineering Experiments
Prototype Experiments
● Medium and Antibiotic
Materials
· Antibiotic
· Double distilled water (ddH2O)
· Tryptone
· NaCl
· Yeast Extract
· Glucose
· Peptone
· H25Na2O16P
· C6H10O8
· Agar
· Absolute ethanol
· Double distilled water (ddH2O)
· Tryptone
· NaCl
· Yeast Extract
· Glucose
· Peptone
· H25Na2O16P
· C6H10O8
· Agar
· Absolute ethanol
Procedure
1.1. Medium:
| Medium | Materials | Mass/g |
| LB Medium (1000mL) | Tryptone | 10 |
| NaCl | 5 | |
| Yeast Extract | 5 | |
| LB Agar (1000mL) | Tryptone | 10 |
| NaCl | 5 | |
| Yeast Extract | 5 | |
| Agar | 15 | |
| HS Medium (500mL) | Glucose | 20 |
| Peptone | 5 | |
| Yeast Extract | 5 | |
| H25Na2O16P | 6.8 | |
| C6H10O8 | 1.5 | |
| HS Agar (500mL) | Glucose | 20 |
| Peptone | 5 | |
| Yeast Extract | 5 | |
| H25Na2O16P | 6.8 | |
| C6H10O8 | 1.5 | |
| Agar | 15 |
1.2. Antibiotic:
1.2.1. Keep the antibiotics until room temperature, 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)
1.2.2. When antibiotics return to room temperature, start weighing clean and dry centrifuge tubes and samples with an analytical balance;
1.2.3. After weighing, use 1mL pipetting gun to suck ultrapure water. Do it faster in front, and slow it down at the last 0.5mL. Stop adding water when the liquid level reaches 10mL;
1.2.4. After dissolution, centrifuge at 5000rpm for 30sec and then suck ultrapure water with a 100μL pipetting gun. After replenishing the liquid level to 10mL again, pass through the membrane to another dry, clean and labeled centrifuge tube and store it at 4℃.
* Note: Chloramphenicol should be dissolved in absolute ethanol.
* Note: Calculation method: Preparation Volume (mL) * Concentration (mg/mL) = Mass (mg)
1.2.2. When antibiotics return to room temperature, start weighing clean and dry centrifuge tubes and samples with an analytical balance;
1.2.3. After weighing, use 1mL pipetting gun to suck ultrapure water. Do it faster in front, and slow it down at the last 0.5mL. Stop adding water when the liquid level reaches 10mL;
1.2.4. After dissolution, centrifuge at 5000rpm for 30sec and then suck ultrapure water with a 100μL pipetting gun. After replenishing the liquid level to 10mL again, pass through the membrane to another dry, clean and labeled centrifuge tube and store it at 4℃.
* Note: Chloramphenicol should be dissolved in absolute ethanol.
● Preparation of Competent Cells
Materials
· Target strain DH5α
· BT medium
· BT Buffer A
· BT Buffer B
· Sterilized LB medium and LB Agar plate
· BT medium
· BT Buffer A
· BT Buffer B
· Sterilized LB medium and LB Agar plate
Procedure
2.1. Take out the bacteria strain from the refrigerator, streak LB plate and culture
it
in a 37℃ incubator.
2.2. Select a monoclonal colony from the plate and inoculate it into the sterilized LB medium, and culture it in a shaker at 37℃, 250rpm for 12h-16h.
2.3. Dilute the bacteria in step 2 into BT medium at the ratio of 1:100, shake the bacteria, and detect OD600 to 0.4-0.7, and the shaking time is about 2-2.5h.
* Note: BT medium shall use conical flasks larger than 250mL, and the shaker shall exceed 250rpm.
2.4. Transfer the bacterial solution to 50mL centrifuge tube and place them on ice for 5-10min.
2.5. Centrifuge at 5000rpm for 5min at 4℃, sip up the waste liquid with pipetting gun and collect precipitation.
2.6. Each 50mL bacterium sediment is re-suspended gently with 16mL ice-precooled BT Buffer A.
2.7. Repeat the step 5.
2.8. Each 50mL bacterium sediment is resuspended gently with 4mL ice-precooled BT Buffer B.
2.9. Each 100μL is individually packed into a 1.5mL centrifuge tube. After packing, fasten down the cover and put it on the ice. When it is almost enough for a box, quickly put it in the box and store it at -80℃.
*Note: All operation must be done on the ice.
2.2. Select a monoclonal colony from the plate and inoculate it into the sterilized LB medium, and culture it in a shaker at 37℃, 250rpm for 12h-16h.
2.3. Dilute the bacteria in step 2 into BT medium at the ratio of 1:100, shake the bacteria, and detect OD600 to 0.4-0.7, and the shaking time is about 2-2.5h.
* Note: BT medium shall use conical flasks larger than 250mL, and the shaker shall exceed 250rpm.
2.4. Transfer the bacterial solution to 50mL centrifuge tube and place them on ice for 5-10min.
2.5. Centrifuge at 5000rpm for 5min at 4℃, sip up the waste liquid with pipetting gun and collect precipitation.
2.6. Each 50mL bacterium sediment is re-suspended gently with 16mL ice-precooled BT Buffer A.
2.7. Repeat the step 5.
2.8. Each 50mL bacterium sediment is resuspended gently with 4mL ice-precooled BT Buffer B.
2.9. Each 100μL is individually packed into a 1.5mL centrifuge tube. After packing, fasten down the cover and put it on the ice. When it is almost enough for a box, quickly put it in the box and store it at -80℃.
*Note: All operation must be done on the ice.
● Preparation of Cellulase:40 mg/mL
Materials
· Cellulase powder
· ddH2O
· ddH2O
Procedure
3.1. Weigh 1g cellulase 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 will affect our final volume calibration.
3.2. Therefore, 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.
3.3. After there is not much foam, calibrate the volume to 25mL and then pass through the membrane.
3.2. Therefore, 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.
3.3. After there is not much foam, calibrate the volume to 25mL and then pass through the membrane.
● Minimum Inhibition Concentration
Materials
· HS medium
· 0.2% cellulase
· Antibiotic
· 0.2% cellulase
· Antibiotic
Procedure
4.1. Gluconacetobacter hansenii ATCC 53582 stored in glycerol is inoculated in a 50mL
centrifuge
tube of
10mL HS medium and 0.2% cellulase, and cultured at 230 rpm, 30℃ for 24-72h until OD600 >
0.7.
4.2. Gluconacetobacter hansenii ATCC 53582 in step 1 is inoculated to 50mL centrifuge tube containing 10mL HS medium and 0.2%(v/v) cellulose, and cultured overnight at 230rpm, 30℃ until OD600= 0.4-0.7. Then centrifuge at 3200rpm for 14min, discard supernatant solution and resuspend with 1mL HS medium.
4.3. The activated Gluconacetobacter hansenii ATCC 53582 is inoculated with a transferring loop and divided into the corresponding antibiotic plate. After culturing at 30℃ for 24-72h, you can observe and record the growth of bacterial plaque.
4.2. Gluconacetobacter hansenii ATCC 53582 in step 1 is inoculated to 50mL centrifuge tube containing 10mL HS medium and 0.2%(v/v) cellulose, and cultured overnight at 230rpm, 30℃ until OD600= 0.4-0.7. Then centrifuge at 3200rpm for 14min, discard supernatant solution and resuspend with 1mL HS medium.
4.3. The activated Gluconacetobacter hansenii ATCC 53582 is inoculated with a transferring loop and divided into the corresponding antibiotic plate. After culturing at 30℃ for 24-72h, you can observe and record the growth of bacterial plaque.
● Plasmid Construction
Materials
· Template and primer
· ddH2O
· Buffer, dNTP, enzyme (it is different between PCR and ligation)
· GelRed
· Binding Buffer and SPW Wash Buffer
· Competent cells
· LB medium and antibiotic
· solution I, solution II, solution III
· HBC Buffer and DNA Wash Buffer
· glycerol
· ddH2O
· Buffer, dNTP, enzyme (it is different between PCR and ligation)
· GelRed
· Binding Buffer and SPW Wash Buffer
· Competent cells
· LB medium and antibiotic
· solution I, solution II, solution III
· HBC Buffer and DNA Wash Buffer
· glycerol
Procedure
5.1. Add 25μL 2 × Phanta Max Buffer, 20μL ddH2O, 1μL dNTP Mix (10 mM each), 1μL Phanta Max
Super-Fidelity DNA Polymerase, 1μL templates (need to dilute to 30ng/μL) and corresponding primers,
oscillate and send them to the PCR thermal cycler (ProFlex 3x32-Well).
5.2. The amplified products were analyzed by 1% agarose gel electrophoresis, compare BP and cut their correct range. Then carry out gel recovery:
5.2.1. Add 300μL Binding Buffer, centrifuge at 13000rpm for 1min, discard waste liquid——repeat twice;
5.2.2. Add 700μL SPW Wash Buffer, centrifuge at 13000rpm for 1min, discard waste liquid——repeat twice;
5.2.3. Centrifuge at 13000rpm for 2min——in order to remove alcohol;
5.2.4. Add 30μL ddH2O and stand for 2 min, centrifuge at 13000rpm for 1min;
5.2.5. Measure the PCR concentration.
5.3. Ligation:
Add 2μL 5×CE Multis Buffer, 1μL enzyme (Exnase Multis), appropriate volume of fragment, carrier and ddH2O. And send them to the PCR thermal cycler.
*Calculation:
(1) The volume of fragment and carrier=(20×BP)/Concentration of PCR;
(2) The volume of ddH2O=10μL-the volume of all solutions expect ddH2O.
5.4. Transformation:
5.4.1. Thaw the competent cells (take ice), add the bacterium liquid ligated and apply ice for 30min. Heat it for 90sec at 42℃ in a water bath, then apply ice again for 2min;
5.4.2. Add 500μL LB to bacterium liquid, shake it at 37℃ and 220 rpm for 1h in a shaker, then centrifuge at 13000rpm for 1 min;
5.4.3. Discard the supernatant solution, resuspend the plasmid with a pipetting gun, take 100μL to the plate (LB and antibiotic), evenly spread with a cell spreader and culture it overnight at 37℃.
5.5. The PCR identification:(If PCR identification is not required, pick the bacteria and spot separately on the plate, compare their growth under blue light and dark conditions)
5.5.1. Pick bacterial plaque, crack at 95℃ for 10min in a water bath and centrifuge at 13000 rpm for 2min;
5.5.2. Take 2μL supernatant solution, add 5μL buffer, 2μL ddH2O,0.25μL dNTP, 0.25μL enzyme and 0.25μL primers, oscillate and send to the PCR thermal cycler.
5.5.3. The amplified products were analyzed by 1% agarose gel electrophoresis, compare BP and record the correct bacterial. Then pick and inoculate the bacterial to medium (including antibiotic), shake them at 37℃ and 250rpm for 12-16h in a shaker.
5.6. Plasmid extraction:
5.6.1. Take 1.5mL bacterial liquid and centrifuge at 13000rpm for 1min, discard waste liquid——repeat three times;
5.6.2. Add 250μL solution I and resuspend; Add 250μL solution II and shake gently; Add 250μL solution III and shake quickly, then centrifuge at 13000rpm for 15min;
5.6.3. Take the supernatant solution to the absorption tube and centrifuge at 13000rpm for 1min, discard the waste liquid;
5.6.4. Add 500μL HBC Buffer and centrifuge at 13000rpm for 1min, discard the waste liquid;
5.6.5. Add 700μL DNA Wash Buffer and centrifuge at 13000rpm for 1min, discard the waste liquid——repeat twice;
5.6.6. Centrifuge at 13000rpm for 2min;
5.6.7. Add 50μL ddH2O, stand for 2min and centrifuge at 13000rpm for 1min;
5.6.8. Measure the concentration.
5.7. Preserve the bacterial liquid to glycerol and send the plasmid to sequence.
5.2. The amplified products were analyzed by 1% agarose gel electrophoresis, compare BP and cut their correct range. Then carry out gel recovery:
5.2.1. Add 300μL Binding Buffer, centrifuge at 13000rpm for 1min, discard waste liquid——repeat twice;
5.2.2. Add 700μL SPW Wash Buffer, centrifuge at 13000rpm for 1min, discard waste liquid——repeat twice;
5.2.3. Centrifuge at 13000rpm for 2min——in order to remove alcohol;
5.2.4. Add 30μL ddH2O and stand for 2 min, centrifuge at 13000rpm for 1min;
5.2.5. Measure the PCR concentration.
5.3. Ligation:
Add 2μL 5×CE Multis Buffer, 1μL enzyme (Exnase Multis), appropriate volume of fragment, carrier and ddH2O. And send them to the PCR thermal cycler.
*Calculation:
(1) The volume of fragment and carrier=(20×BP)/Concentration of PCR;
(2) The volume of ddH2O=10μL-the volume of all solutions expect ddH2O.
5.4. Transformation:
5.4.1. Thaw the competent cells (take ice), add the bacterium liquid ligated and apply ice for 30min. Heat it for 90sec at 42℃ in a water bath, then apply ice again for 2min;
5.4.2. Add 500μL LB to bacterium liquid, shake it at 37℃ and 220 rpm for 1h in a shaker, then centrifuge at 13000rpm for 1 min;
5.4.3. Discard the supernatant solution, resuspend the plasmid with a pipetting gun, take 100μL to the plate (LB and antibiotic), evenly spread with a cell spreader and culture it overnight at 37℃.
5.5. The PCR identification:(If PCR identification is not required, pick the bacteria and spot separately on the plate, compare their growth under blue light and dark conditions)
5.5.1. Pick bacterial plaque, crack at 95℃ for 10min in a water bath and centrifuge at 13000 rpm for 2min;
5.5.2. Take 2μL supernatant solution, add 5μL buffer, 2μL ddH2O,0.25μL dNTP, 0.25μL enzyme and 0.25μL primers, oscillate and send to the PCR thermal cycler.
5.5.3. The amplified products were analyzed by 1% agarose gel electrophoresis, compare BP and record the correct bacterial. Then pick and inoculate the bacterial to medium (including antibiotic), shake them at 37℃ and 250rpm for 12-16h in a shaker.
5.6. Plasmid extraction:
5.6.1. Take 1.5mL bacterial liquid and centrifuge at 13000rpm for 1min, discard waste liquid——repeat three times;
5.6.2. Add 250μL solution I and resuspend; Add 250μL solution II and shake gently; Add 250μL solution III and shake quickly, then centrifuge at 13000rpm for 15min;
5.6.3. Take the supernatant solution to the absorption tube and centrifuge at 13000rpm for 1min, discard the waste liquid;
5.6.4. Add 500μL HBC Buffer and centrifuge at 13000rpm for 1min, discard the waste liquid;
5.6.5. Add 700μL DNA Wash Buffer and centrifuge at 13000rpm for 1min, discard the waste liquid——repeat twice;
5.6.6. Centrifuge at 13000rpm for 2min;
5.6.7. Add 50μL ddH2O, stand for 2min and centrifuge at 13000rpm for 1min;
5.6.8. Measure the concentration.
5.7. Preserve the bacterial liquid to glycerol and send the plasmid to sequence.
● Electrotransformation
Materials
· HS medium + 0.2% cellulase
· 1mM HEPES buffer
· 15% glycerol
· HS medium
· HS Agar plate (including antibiotic)
· 1mM HEPES buffer
· 15% glycerol
· HS medium
· HS Agar plate (including antibiotic)
Procedure
6.1. Inoculate Gluconacetobacter hansenii ATCC 53582 to 50mL centrifuge tube of 5mL HS medium
and
0.2%(v/v) cellulase, shake it at 30℃ and 180rpm for 24-72h in a shaker until OD600>0.7.
6.2. Inoculate to 10mL HS medium and 0.2% cellulase in a 250mL conical flask, shake it at 180rpm and 30℃ for overnight until OD600=0.4-0.7.
6.3. Precool centrifuge to 4℃, prepare ice bucket and ice and put 1mM HEPES buffer and 15% glycerol into the ice.
6.4. Take out the reaching OD600 bacterial to ice for 10min.
6.5. Centrifuge at 4100rpm and 4℃ for 12min, discard the supernatant solution.
*Note: Pour out the supernatant solution carefully but not the particles. Gluconacetobacter hansenii ATCC 53582 cannot form pellet as easily as Escherichia coli (E.coli), most likely due to the buffering effect of cellulase. If the pellet is not attached to the tube wall after centrifuging, the pellet is smeared on the wall and centrifuged for a long time. After pouring the supernatant solution, gently sip it up or buckle on the tissue paper.
6.6. Resuspend with 10mL HEPES buffer.
*Note: Firstly resuspend with 1mL HEPES buffer, then add 9mL HEPES buffer with an electric pipetting gun.
6.7. Centrifuge at 4100rpm and 4℃ for 12min, discard the supernatant solution. Repeat steps 6.5. to 6.7.
6.8. After resuspending with 5mL 15% glycerol, place in a 4℃ centrifuge, 4100rpm, 14min and discard the supernatant solution.
6.9. After resuspending particles with 1mL 15% glycerol (glacial), each 100μL is packed into 1.5mL centrifuge tubes individually.
*Note: After freezing, the efficiency of the active battery may decrease, so immediate use may produce the highest efficiency. 100μL, that is, the volume of plasmid plus Gluconacetobacter hansenii ATCC 53582 is 100μL, and the final resuspension volume depends on the number of samples.
6.10. Add 600ng plasmid to competent cells and gently flick, then suck it into (0.1cm) electroporation cup and set 3000V and 5-8ms. After finishing, take immediately the bacterial to 500mL HS medium and cellulase, and culture it at 180rpm and 30℃ for 16h. Calculation: the volume of plasmid=600ng/Concentration (no more than 10μL.)
6.11. After centrifuging for 1min, resuspend with 100μL medium and spread the HS Agar (including antibiotic), the colony will grow plaque in 24-72h. It needs to do the PCR identification to ensure the correct plasmid.
6.2. Inoculate to 10mL HS medium and 0.2% cellulase in a 250mL conical flask, shake it at 180rpm and 30℃ for overnight until OD600=0.4-0.7.
6.3. Precool centrifuge to 4℃, prepare ice bucket and ice and put 1mM HEPES buffer and 15% glycerol into the ice.
6.4. Take out the reaching OD600 bacterial to ice for 10min.
6.5. Centrifuge at 4100rpm and 4℃ for 12min, discard the supernatant solution.
*Note: Pour out the supernatant solution carefully but not the particles. Gluconacetobacter hansenii ATCC 53582 cannot form pellet as easily as Escherichia coli (E.coli), most likely due to the buffering effect of cellulase. If the pellet is not attached to the tube wall after centrifuging, the pellet is smeared on the wall and centrifuged for a long time. After pouring the supernatant solution, gently sip it up or buckle on the tissue paper.
6.6. Resuspend with 10mL HEPES buffer.
*Note: Firstly resuspend with 1mL HEPES buffer, then add 9mL HEPES buffer with an electric pipetting gun.
6.7. Centrifuge at 4100rpm and 4℃ for 12min, discard the supernatant solution. Repeat steps 6.5. to 6.7.
6.8. After resuspending with 5mL 15% glycerol, place in a 4℃ centrifuge, 4100rpm, 14min and discard the supernatant solution.
6.9. After resuspending particles with 1mL 15% glycerol (glacial), each 100μL is packed into 1.5mL centrifuge tubes individually.
*Note: After freezing, the efficiency of the active battery may decrease, so immediate use may produce the highest efficiency. 100μL, that is, the volume of plasmid plus Gluconacetobacter hansenii ATCC 53582 is 100μL, and the final resuspension volume depends on the number of samples.
6.10. Add 600ng plasmid to competent cells and gently flick, then suck it into (0.1cm) electroporation cup and set 3000V and 5-8ms. After finishing, take immediately the bacterial to 500mL HS medium and cellulase, and culture it at 180rpm and 30℃ for 16h. Calculation: the volume of plasmid=600ng/Concentration (no more than 10μL.)
6.11. After centrifuging for 1min, resuspend with 100μL medium and spread the HS Agar (including antibiotic), the colony will grow plaque in 24-72h. It needs to do the PCR identification to ensure the correct plasmid.
● The Inhibition Zone Experiment of SE Protein
Materials
· LB Agar plate
· FAB medium
· Pseudomonas aeruginosa (PAO1)
· 1mM FeCl3
· 1mol glutamate
· FAB medium+1% Agar
· FAB medium
· Pseudomonas aeruginosa (PAO1)
· 1mM FeCl3
· 1mol glutamate
· FAB medium+1% Agar
Procedure
7.1. Streak the PAO1 to LB Agar plate.
7.2. Select the monoclonal point and inoculate to FAB medium, FeCl3, glutamate (each 1mL FAB medium+30μL 1mol glutamate+1μL 1mM FeCl3, the same as below) in a 250mL conical flask and shake it until OD600=0.5-0.8.
*Calculation:
(1) OD1×V1=OD2×V2(OD1—data measured, V1—data required, OD2—5, V2—volume required)
(2) V2=V requirement +V loss
7.3. Collect the bacterial(V1) from step 2 with 50mL centrifuge tube, centrifuge at 4000rpm for 5min and discard the waste liquid.
7.4. Resuspend with FAB medium(V2), glutamate and FeCl3.
7.5. Suck the volume required to other 50mL centrifuge tube, add FAB medium+1% Agar, FeCl3, glutamate (the total volume of each plate is 5mL), mix, pour it in a plate and blow for 45min.
*Note: When OD600=0.5, the bacterial number is 109, the volume required is 2mL; When OD600=5, the bacterial number is 5×109, the volume required is 1mL.
7.6. Drip 3μL supernatant solution to the plate and observe the growth of the inhibition zone.
7.2. Select the monoclonal point and inoculate to FAB medium, FeCl3, glutamate (each 1mL FAB medium+30μL 1mol glutamate+1μL 1mM FeCl3, the same as below) in a 250mL conical flask and shake it until OD600=0.5-0.8.
*Calculation:
(1) OD1×V1=OD2×V2(OD1—data measured, V1—data required, OD2—5, V2—volume required)
(2) V2=V requirement +V loss
7.3. Collect the bacterial(V1) from step 2 with 50mL centrifuge tube, centrifuge at 4000rpm for 5min and discard the waste liquid.
7.4. Resuspend with FAB medium(V2), glutamate and FeCl3.
7.5. Suck the volume required to other 50mL centrifuge tube, add FAB medium+1% Agar, FeCl3, glutamate (the total volume of each plate is 5mL), mix, pour it in a plate and blow for 45min.
*Note: When OD600=0.5, the bacterial number is 109, the volume required is 2mL; When OD600=5, the bacterial number is 5×109, the volume required is 1mL.
7.6. Drip 3μL supernatant solution to the plate and observe the growth of the inhibition zone.
● Expression and Identification of SE Antibacterial
Protein of
Gluconacetobacter hansenii ATCC 53582
Materials
· 1×PBS buffer
· PMSF
· 1 mg/mL lysozyme
· HS medium+cellulase+Chl148
· 4×Protein Loading Buffer
· Coomassie brilliant blue
· PMSF
· 1 mg/mL lysozyme
· HS medium+cellulase+Chl148
· 4×Protein Loading Buffer
· Coomassie brilliant blue
Procedure
8.1. Shake the Gluconacetobacter hansenii ATCC 53582 with different expression levels of correct SE.
8.2. Inoculate the bacterial (step 1) to HS medium+cellulase+Chl148(the ratio of 1:50), shake the bacterial until logarithmic phase (0.3-0.7).
8.3. Pack individually the bacterial liquid to 50mL centrifuge tube, centrifuge at 4000rpm for 5min.
8.4. Wash the bacterial with 1×PBS buffer and centrifuge at 4000rpm for 5min, discard the supernatnt solution.
8.5. Add 5mL 1×PBS buffer (corresponding 200mL bacterial liquid) and put it in -80℃ refrigerator freeze-thaw once.
8.6. Take out the bacterial liquid from refrigerator and add 10μL PMSF, 1 mg/mL lysozyme, then heat it at 37℃ in a water bath.
8.7. Ultrasonic wave: Power is 50% (about 400W), working time is 1sec, interval time is 3sec, and total working time is 3min.
8.8. Centrifuge(high-speed) at 8000rpm and 4℃ for 20min.
8.9. Take the supernatant solution to pass through the membrane, then put the samples to 4℃ refrigerator.
8.10. Measure the absorbance of protein solution of collection tube at 280nm, estimate roughly the concentration of samples.
8.11. Take the high concentration of several tubes of protein samples and control group bacterial suspension, add 4×Protein Loading Buffer, put it at 98℃ for 5-8min and make it fully denatured. Make SDS-PAGE electrophoresis, set 100V for about 2h to make bromophenol blue run to the bottom.
8.12. Coomassie brilliant blue dyeing for 30min.
8.13. Decolorize for overnight, or heat in the microwave.
8.14. Take some photos and judge the concentration and purity of the target protein.
8.2. Inoculate the bacterial (step 1) to HS medium+cellulase+Chl148(the ratio of 1:50), shake the bacterial until logarithmic phase (0.3-0.7).
8.3. Pack individually the bacterial liquid to 50mL centrifuge tube, centrifuge at 4000rpm for 5min.
8.4. Wash the bacterial with 1×PBS buffer and centrifuge at 4000rpm for 5min, discard the supernatnt solution.
8.5. Add 5mL 1×PBS buffer (corresponding 200mL bacterial liquid) and put it in -80℃ refrigerator freeze-thaw once.
8.6. Take out the bacterial liquid from refrigerator and add 10μL PMSF, 1 mg/mL lysozyme, then heat it at 37℃ in a water bath.
8.7. Ultrasonic wave: Power is 50% (about 400W), working time is 1sec, interval time is 3sec, and total working time is 3min.
8.8. Centrifuge(high-speed) at 8000rpm and 4℃ for 20min.
8.9. Take the supernatant solution to pass through the membrane, then put the samples to 4℃ refrigerator.
8.10. Measure the absorbance of protein solution of collection tube at 280nm, estimate roughly the concentration of samples.
8.11. Take the high concentration of several tubes of protein samples and control group bacterial suspension, add 4×Protein Loading Buffer, put it at 98℃ for 5-8min and make it fully denatured. Make SDS-PAGE electrophoresis, set 100V for about 2h to make bromophenol blue run to the bottom.
8.12. Coomassie brilliant blue dyeing for 30min.
8.13. Decolorize for overnight, or heat in the microwave.
8.14. Take some photos and judge the concentration and purity of the target protein.
● The Verification Experiment of BC Film Yield on
12-Well Cell Culture plates
Materials
· HS medium
· Cellulase
· Chl148
· ddH2O
· 0.9% NaCl
· Cellulase
· Chl148
· ddH2O
· 0.9% NaCl
Procedure
9.1. After electrotransformation, spread the bacterial to the plate with a cell
spreader.
Select a monoclonal point to shake the bacterial with HS medium+Chl148+cellulase.
9.2. Shake until the stable phase and detect the OD600>0.8.
9.3. Dilute the stable phase bacterial liquid at the ratio of 1:75 to shake with a 10mL conical flask, detect the OD600=0.2-0.8.
9.4. Before experiment, measure the final OD600 of each bacterium.
9.5. Calculate the OD600 of the final obtained volume of each bacterium is 0.5.
9.5.1. V obtainment=V HS medium + V corresponding antibiotic: the number of conditions × the number of samples × 1.6mL (the volume required for each group), you need to calculate the volume of loss.
9.5.2. OD resuspension=0.5: HS medium + corresponding volume/25(dilution)
9.5.3. OD aim × V aim=OD measurement × V obtainment
9.6. After collecting the bacterial, suck up the supernatant solution with pipetting gun.
9.7. Resuspend with 1mL 0.9% NaCl solution.
9.8. Centrifuge at 10000rpm for 3min.
9.9. Repeat steps 7 and 8.
9.10. Resuspend with 1mL 0.9% NaCl solution and unify all the bacterial until OD600=0.5.
9.11. Dilute the OD600=0.5 resuspension at the ratio of 1:25 to original solution (HS medium+ corresponding antibiotic) and mix.
9.12. Suck accurately 3mL the mixture solution to 12-well cell culture plate and make three parallel samples of each bacterium under the same condition.
9.13. Culture stationarily at 30℃ for 16h under the red light and dark conditions.
*Note: The bacterial must to sip up to ensure the variables of the volume, culture time, temperature (and so on) unchanged and the red light is a single variable.
9.2. Shake until the stable phase and detect the OD600>0.8.
9.3. Dilute the stable phase bacterial liquid at the ratio of 1:75 to shake with a 10mL conical flask, detect the OD600=0.2-0.8.
9.4. Before experiment, measure the final OD600 of each bacterium.
9.5. Calculate the OD600 of the final obtained volume of each bacterium is 0.5.
9.5.1. V obtainment=V HS medium + V corresponding antibiotic: the number of conditions × the number of samples × 1.6mL (the volume required for each group), you need to calculate the volume of loss.
9.5.2. OD resuspension=0.5: HS medium + corresponding volume/25(dilution)
9.5.3. OD aim × V aim=OD measurement × V obtainment
9.6. After collecting the bacterial, suck up the supernatant solution with pipetting gun.
9.7. Resuspend with 1mL 0.9% NaCl solution.
9.8. Centrifuge at 10000rpm for 3min.
9.9. Repeat steps 7 and 8.
9.10. Resuspend with 1mL 0.9% NaCl solution and unify all the bacterial until OD600=0.5.
9.11. Dilute the OD600=0.5 resuspension at the ratio of 1:25 to original solution (HS medium+ corresponding antibiotic) and mix.
9.12. Suck accurately 3mL the mixture solution to 12-well cell culture plate and make three parallel samples of each bacterium under the same condition.
9.13. Culture stationarily at 30℃ for 16h under the red light and dark conditions.
*Note: The bacterial must to sip up to ensure the variables of the volume, culture time, temperature (and so on) unchanged and the red light is a single variable.
● The Treatment of BC Film(Drying Method)
Materials
· 100mM NaOH solution
· ddH2O
· ddH2O
Procedure
10.1. Put three parallels under the same condition to the same beaker with a tweezer, and label the
culture condition to avoid the confusion.
10.2. Shake and wash for 5min with ddH2O, repeat three times.
10.3. Soak in 100mM NaOH solution and boil until the BC film become transparent.
10.4. Shake and wash for 5min with ddH2O, repeat three times.
10.5. Place each membrane to a single beaker and label the culture condition.
10.6. Put them into 85℃ oven until constant weight.
*Note: Weighting after beaker are cooled to room temperature.
10.7. m constant weight - m beaker =m membrane.
10.2. Shake and wash for 5min with ddH2O, repeat three times.
10.3. Soak in 100mM NaOH solution and boil until the BC film become transparent.
10.4. Shake and wash for 5min with ddH2O, repeat three times.
10.5. Place each membrane to a single beaker and label the culture condition.
10.6. Put them into 85℃ oven until constant weight.
*Note: Weighting after beaker are cooled to room temperature.
10.7. m constant weight - m beaker =m membrane.
● Quantitative Fluorescence Intensity
Materials
· LB medium
Procedure
11.1. Shake the bacterial with LB medium for 12-16h.
11.2. Dilute the bacterial at the ratio of 1:100 to shake until OD600=0.2(about), and dilute them at the bacterial at the ratio of 1:10 to shake until OD600=0.2(about).
11.3. Unify all bacterial OD600 to 2, suck 180μL of each sample to 96-well cell culture plates and do three parallel samples.
11.4. Measure three green fluorescence intensity and OD600.
11.2. Dilute the bacterial at the ratio of 1:100 to shake until OD600=0.2(about), and dilute them at the bacterial at the ratio of 1:10 to shake until OD600=0.2(about).
11.3. Unify all bacterial OD600 to 2, suck 180μL of each sample to 96-well cell culture plates and do three parallel samples.
11.4. Measure three green fluorescence intensity and OD600.
Prototype Experiments
● The Experiment of Freeze-drying Bacterial
Materials
· Gluconacetobacter hansenii ATCC 53582 and the bacterial are modified by synthetic biology
· Dry skim milk (WONDERSUN BRAND)
· HS medium and HS Agar
· PBS buffer
.Physio logical saline
· Dry skim milk (WONDERSUN BRAND)
· HS medium and HS Agar
· PBS buffer
.Physio logical saline
Procedure
1.1. Oscillate the conical flask containing the bacteria that can produce the BC film on the vortex
mixer for 3-4min to shake the bacteria out of the film.
1.2. Suck up 1mL bacterial liquid from the standing conical flask to inoculate to 100mL HS medium, shake it at 180rpm and 30℃ until OD600=0.8.
1.3. Suck up 3mL from bacterial liquid to 50mL centrifuge tube, centrifuge at 4100rpm for 12min, discard quickly the supernatant solution and collect the bacterial.
1.4. Wash twice with PBS buffer (pH is 7.2-7.4), and centrifuge to collect.
1.5. Add 3mL protective agent (10% dry skim milk) and phosphoric acid buffer, and mix fully with the vortex mixer.
1.6. After pre-freezing at -80℃ for 3h, put the bacterial tubes to freezing box and cover with tin- foil, then put them into lyophilizer.
*Note: You need to detect engine oil before using the lyophilizer, then use it.
1.7. After freezing at -80℃ for 24h, remove the centrifuge tubes from lyophilizer.
*Note: You need to wait until the lyophilizer gets down to -40℃ then you can leave.
1.8. Pour the plates on the alternate days. Blow for 30min and UV irradiate for 30min.
1.9. Before putting the centrifuge tube in the super clean bath, wipe 50mL centrifuge tube wall with 70% alcohol and resuspend with 3mL PBS buffer.
1.10. The determination of bacterial survival rate:
1.10.1. Before processing:
1.10.1.1. After shaking fully the liquid samples, suck to 25mL samples with sterile suction pipe and put into the sterile conical flask containing 225mL physio logical saline (place the appropriate amount of sterile glass beads), shake fully to make 1:10 sample solution.
1.10.1.2. Suck 1:10 sample solution to sterile test tube containing 9mL physiological saline (add slowly along the tube wall and the tip does not touch the dilution solution), shake the test tube to mix fully to make 1:100 sample solution.
1.10.1.3. Take another 1mL micropipette sucker. According to the above-mentioned operation, make 10 times increment sample solution. Replace 1mL sterile sucker with 1 dilution per increment. Make a total of 8 gradients.
1.10.1.4. Select 2-3 consecutive appropriate dilutions, suck 100μL sample solution of each dilution to culture plate to spread (5 plates per dilution) and affix the seal, culture at 30℃ for 48h in the constant temperature incubator.
1.10.2. After resuspending:
1.10.2.1. Suck up 1mL sample solution with 1mL micropipette and add to sterile test tube containing 9mL physiological saline (add slowly along the tube wall and the tip does not touch the dilution solution), shake the test tube to mix fully to make 1:10 sample solution.
1.10.2.2. Take another 1mL micropipette sucker. According to the above-mentioned operation, make 10 times increment sample solution. Replace 1mL sterile sucker with 1 dilution per increment. Make a total of 4 gradients.
1.10.2.3. Select 2-3 consecutive appropriate dilutions, suck 100μL sample solution of each dilution to culture plate to spread (5 plates per dilution) and affix the seal, culture at 30℃ for 48h in the constant temperature incubator.
1.11. Calculation:
1.11.1. Select the colony with a CFU between 30 and 300 without spreading colony growth, and measure the total number of colonies by plate counting method. If it is lower than 30CFU, record the specific number of colonies by plate counting method. If it is larger than 300CFU, it can be recorded as too many to count. The number of colonies per dilution should be averaged on two plate numbers.
1.11.2. Report on the number of colonies: When the number of colonies is less than 100CFU, according to a “rounding” principle revision, take the first two digits and replace them with 0. It is also expressed by the exponential form of 10 and then according to the a “rounding” principle revision, two digits valid numbers shall be adopted.
1.2. Suck up 1mL bacterial liquid from the standing conical flask to inoculate to 100mL HS medium, shake it at 180rpm and 30℃ until OD600=0.8.
1.3. Suck up 3mL from bacterial liquid to 50mL centrifuge tube, centrifuge at 4100rpm for 12min, discard quickly the supernatant solution and collect the bacterial.
1.4. Wash twice with PBS buffer (pH is 7.2-7.4), and centrifuge to collect.
1.5. Add 3mL protective agent (10% dry skim milk) and phosphoric acid buffer, and mix fully with the vortex mixer.
1.6. After pre-freezing at -80℃ for 3h, put the bacterial tubes to freezing box and cover with tin- foil, then put them into lyophilizer.
*Note: You need to detect engine oil before using the lyophilizer, then use it.
1.7. After freezing at -80℃ for 24h, remove the centrifuge tubes from lyophilizer.
*Note: You need to wait until the lyophilizer gets down to -40℃ then you can leave.
1.8. Pour the plates on the alternate days. Blow for 30min and UV irradiate for 30min.
1.9. Before putting the centrifuge tube in the super clean bath, wipe 50mL centrifuge tube wall with 70% alcohol and resuspend with 3mL PBS buffer.
1.10. The determination of bacterial survival rate:
1.10.1. Before processing:
1.10.1.1. After shaking fully the liquid samples, suck to 25mL samples with sterile suction pipe and put into the sterile conical flask containing 225mL physio logical saline (place the appropriate amount of sterile glass beads), shake fully to make 1:10 sample solution.
1.10.1.2. Suck 1:10 sample solution to sterile test tube containing 9mL physiological saline (add slowly along the tube wall and the tip does not touch the dilution solution), shake the test tube to mix fully to make 1:100 sample solution.
1.10.1.3. Take another 1mL micropipette sucker. According to the above-mentioned operation, make 10 times increment sample solution. Replace 1mL sterile sucker with 1 dilution per increment. Make a total of 8 gradients.
1.10.1.4. Select 2-3 consecutive appropriate dilutions, suck 100μL sample solution of each dilution to culture plate to spread (5 plates per dilution) and affix the seal, culture at 30℃ for 48h in the constant temperature incubator.
1.10.2. After resuspending:
1.10.2.1. Suck up 1mL sample solution with 1mL micropipette and add to sterile test tube containing 9mL physiological saline (add slowly along the tube wall and the tip does not touch the dilution solution), shake the test tube to mix fully to make 1:10 sample solution.
1.10.2.2. Take another 1mL micropipette sucker. According to the above-mentioned operation, make 10 times increment sample solution. Replace 1mL sterile sucker with 1 dilution per increment. Make a total of 4 gradients.
1.10.2.3. Select 2-3 consecutive appropriate dilutions, suck 100μL sample solution of each dilution to culture plate to spread (5 plates per dilution) and affix the seal, culture at 30℃ for 48h in the constant temperature incubator.
1.11. Calculation:
1.11.1. Select the colony with a CFU between 30 and 300 without spreading colony growth, and measure the total number of colonies by plate counting method. If it is lower than 30CFU, record the specific number of colonies by plate counting method. If it is larger than 300CFU, it can be recorded as too many to count. The number of colonies per dilution should be averaged on two plate numbers.
1.11.2. Report on the number of colonies: When the number of colonies is less than 100CFU, according to a “rounding” principle revision, take the first two digits and replace them with 0. It is also expressed by the exponential form of 10 and then according to the a “rounding” principle revision, two digits valid numbers shall be adopted.
The bacterial survival rate/%=
● The Experiment of Freeze-drying Bacterial
Materials
· Gluconacetobacter hansenii ATCC 53582 and the bacterial are modified by synthetic
biology
· Tween-80,Tween-20
· EHP
· Hydrogenated polyisobutene
· Sepigel 305
· HS medium
· Sterile water
· Tween-80,Tween-20
· EHP
· Hydrogenated polyisobutene
· Sepigel 305
· HS medium
· Sterile water
Procedure
2.1.Oscillate the conical flask containing the bacteria that can produce the BC film on the vortex
mixer
for 3-4min to shake the bacteria out of the film.
2.2.Suck up 1mL bacterial liquid from the standing conical flask to inoculate to 100mL HS medium, shake at 180rpm and 30℃ until OD600=0.5-0.8.
2.3.The obtained volume OD600 is 3, based on the formula: c measure × V measure=c aim × V aim.
2.4.Suck 3mL culture liquid to 50mL centrifuge tube, centrifuge at 4100rpm for 12min, discard quickly the supernatant solution and collect the bacterial.
2.5.Wash twice with sterile water and centrifuge to collect.
2.6.Add the volume of aim medium and corresponding antibiotic, resuspend and mix.
2.7.Under the sterile condition, add aqueous phase, surfactant and oil phase. After oscillating and emulsify until becomes homogeneous phase, add thickener and shake fully to make it dissolve.
*Note: If there is so much foam,you can centrifuge at 300rpm for 5min.
2.8.Under the red light and dark conditions,take 1g emulsion to 35mm culture plate with electronic balance.
2.2.Suck up 1mL bacterial liquid from the standing conical flask to inoculate to 100mL HS medium, shake at 180rpm and 30℃ until OD600=0.5-0.8.
2.3.The obtained volume OD600 is 3, based on the formula: c measure × V measure=c aim × V aim.
2.4.Suck 3mL culture liquid to 50mL centrifuge tube, centrifuge at 4100rpm for 12min, discard quickly the supernatant solution and collect the bacterial.
2.5.Wash twice with sterile water and centrifuge to collect.
2.6.Add the volume of aim medium and corresponding antibiotic, resuspend and mix.
2.7.Under the sterile condition, add aqueous phase, surfactant and oil phase. After oscillating and emulsify until becomes homogeneous phase, add thickener and shake fully to make it dissolve.
*Note: If there is so much foam,you can centrifuge at 300rpm for 5min.
2.8.Under the red light and dark conditions,take 1g emulsion to 35mm culture plate with electronic balance.
References
[1] Yang Jiaping, Li Zhixi, Jiang Xiaozhi, etc. Study on freeze-drying conditions of
acetic
acid bacteria strains [ J ]. Chinese brewing, 2008, 20: 49-52.
[2] Wang Na, Guo Shilei, Zhang Yongxiang, etc. Selection of cryoprotectants for acetic acid bacteria [ J ].Food and fermentation industry, 2015, 41 ( 003 ): 135-139.
[2] Wang Na, Guo Shilei, Zhang Yongxiang, etc. Selection of cryoprotectants for acetic acid bacteria [ J ].Food and fermentation industry, 2015, 41 ( 003 ): 135-139.