Team:USTC/Protocals
Plasmid Extraction:
1. Centrifuge 1.5 mL bacterium solution at 11000 rpm for 2 minutes. Remove the supernatant. Repeat twice.
2. Add 250 μL Buffer P1, resuspend cells.
3. Add 250 μL Buffer P2, mix well, 3 min's standing.
4. Add 350 μL Buffer P3, mix well.
5.13400 rpm centrifuge for 10 min, move all supernatant to adsorption column, 11000 rpm centrifuge 30 s, discard filtrate.
6. Add 500 μL Buffer DW1, 12000 rpm centrifuge 30 s, discard filtrate.
7. Add 500 μL Wash Solution, 12000 rpm centrifuge 30 s, discard filtrate. Repeat once.
8.12000 rpm centrifuge for 1 min.
9. Lying for 10 min.（drying for 3 min）
10. Put the adsorption column in a new EP tube. Add 50 μL 50°C ddH2O, 10 min's standing (3 min’s drying), 12000 rpm centrifuge 1 min. Preserve in the EP tube with ddH2O at 4 degree Celsius.
PCR:
1.Prepare 6 PCR tubes and sequentially add：
| sample | 1 | 3 | 5 | 6 | 8 |
|---|---|---|---|---|---|
| Sterilized ddH2O | 7 μL | 7 μL | 7 μL | 7 μL | 7 μL |
| 2×Primer Star | 10 μL | 10 μL | 10 μL | 10 μL | 10 μL |
| template | 1 μL | 1 μL | 1 μL | 1 μL | 1 μL |
| VF2(10 μM) | 1 μL | 1 μL | 1 μL | 1 μL | 1 μL |
| VR(10 μM) | 1 μL | 1 μL | 1 μL | 1 μL | 1 μL |
| total | 20 μL | 20 μL | 20 μL | 20 μL | 20 μL |
2.PCR reaction Parameters setting：
| stage | temperature | time |
|---|---|---|
| step 1 | 95 | 5 min |
| step 2 | 95 | 20 s |
| step 3 | 55 | 20 s |
| step 4 | 72 | 30 s |
| step 5 | 72 | 10 min |
| step 6 | 4 | -- |
30 cycles(step 2 ~ step 4)
3.Agarose gel electrophoresis mixed with 6× DNA loading buffer;110 V, 30 min Agarose gel
Purification of PCR product:
1. Add 225 μL Buffer B3 to the 25 μL solution and mix it up. Add it to an adsorption column.
2. 11,000 rpm centrifuge for 30 s, and then discard the filtrate.
3. Add 500 μL Wash Solution, 12,000 rpm centrifuge for 30 s, and then discard the filtrate.
4. Repeat last process.
5. Centrifuge the empty column at 12,000 rpm for 1 min.
6. Lie the column still for 10 min.
7. Put the column to an 1.5 ml EP tube, add 20 μL ddH2O, stand them still for 10 min. Centrifuge at 12,000 rpm for 1 min.
Gel Extraction:
1. Cut off the Gel part containing the target band.
2. Weigh the cut off Gel part.
3. Add Buffer B2 which 300 times the weight of the Gel.
4. Add isopropanol which is 1/3 volume of the Buffer B2.
5. Move the solution to adsorption column, 11000 rpm centrifuge 30 s, discard filtrate.
6. Add 500 μL Wash Solution, 12000 rpm centrifuge 30 s, discard filtrate. Repeat once.
7. 12000 rpm centrifuge 1 min.
8. Lying for 10 min.
9. Put the adsorption column in a new EP tube. Add 20 μL ddH2O, 10 min's standing, 12000 rpm centrifuge 1 min. Preserve in the EP tube with ddH2O at 4 degree Celsius.
Transformation:
NOTE: Generally, competent bacteria are restrored in -70 degree centigrade environment.
1. Take the competent bacteria from refrigerator and incubate them into ice about 5 mins until it is dissolved
2. Absorb 100pg to 10 ng plasmid(normally 1 to 2 uL, DO NOT add more than 5% volumn of bacteria solution) and mix it with bacteria solution thoroughly. ATTENTION: Please operate this step tenderly!!!
3. Put the tubes on the ice about 30 mins.(Time SHOULD BE ACCURATE)
4. Make a heat shock at 42 degree centigrade about 90 sec(TIME SHOULD BE ACCURATE)
5. Put the tubes on the ice about 5 mins again.
6. Add 900 ul LB medium into EP tubes and cultivate the bacteria at 37 degree centigrade about 60 min.
7. Centrifuge them at 4000 rpm for 2 minutes and we will see sediment in the tubes.
8. Discard the supernatant liquid and leave about 200 ul medium.
9. Coat plate: Add 200 ul solution in a plate with kanamycin.
10. Cultivate these bacteria overnight for further use.
Colony PCR/Bacteria PCR:
1.Prepare 6 PCR tubes and sequentially add：
| sample | 1 | 3 | 5 | 6 | 8 |
|---|---|---|---|---|---|
| Sterilized ddH2O | 7 μL | 7 μL | 7 μL | 7 μL | 7 μL |
| 2×Primer Star | 10 μL | 10 μL | 10 μL | 10 μL | 10 μL |
| Bacteria | 1 μL | 1 μL | 1 μL | 1 μL | 1 μL |
| VF2(10 μM) | 1 μL | 1 μL | 1 μL | 1 μL | 1 μL |
| VR(10 μM) | 1 μL | 1 μL | 1 μL | 1 μL | 1 μL |
| total | 20 μL | 20 μL | 20 μL | 20 μL | 20 μL |
2.PCR reaction Parameters setting：
| stage | temperature | time |
|---|---|---|
| step 1 | 95 | 5 min |
| step 2 | 95 | 20 s |
| step 3 | 55 | 20 s |
| step 4 | 72 | 30 s |
| step 5 | 72 | 10 min |
| step 6 | 4 | -- |
30 cycles(step 2 ~ step 4)
3.Agarose gel electrophoresis mixed with 6× DNA loading buffer;110 V, 30 min Agarose gel
| Name | Supplier |
|---|---|
| Chloramphenicol | BBI Life Sciences |
| Kanamycin sulfate | BBI Life Sciences |
| Ampicillin | BBI Life Sciences |
Transformation of competent cells
1.Take the competent bacteria from refrigerator and incubate them into ice about 5 mins until it is
dissolved
2.Absorb 100pg to 10 ng plasmid and mix it with bacteria solution thoroughly.
ATTENTION: Please operate this step tenderly!!!
3.Put the tubes on the ice about 30 mins.(Time SHOULD BE ACCURATE)
4.Make a heat shock at 42 degree centigrade about 90 sec (TIME SHOULD BE ACCURATE)
5.Put the tubes on the ice about 5 mins again.
6.Add 900 ul LB medium into EP tubes and cultivate the bacteria at 37 degree centrigrade about
1 hours .
7.Centrifuge them at 4000 rpm about 2 mins and we will see sediment in the tubes.
8.Discard the supernatant liquid and leave about 200 ul medium.
9.Coat plate: add 200 ul solution in a large plate.
10.Cultivate these bacteria overnight for further use.
Preparation of CaCl2 competent cells
1.Pick up some bacterial solution with the inoculating loop and inoculate it onto the anti-LB plate
by plate scribing.
2.Incubate at 37⁰C overnight.
3.Pick colonies in 5 mL EP tubes without anti-LB liquid mediumIncubate at 37⁰C, shaking at
250rpm for 6 hours.
4.Transfer 2 mL of bacterial solution to 200 mL of LB liquid medium at 37 ° C shaker until the
OD value reaches 0.4.
5.Transfer the medium to a 50 mL pre-cooled centrifuge tube and place on ice for 30 min.
6.Spin down for 15 minutes at 4000rpm at 4⁰C.
7.Discard supernatant, resuspend pellet in 30ml of 80mM CaCl2-20mM MgCl2, keep on ice.
8.Spin down for 15 minutes at 2700rpm at 4⁰C.
9.Discard supernatant, resuspend pellet in 2mL 100mM CaCl2-18% glycerin, keep on ice，then
dispense to EP tube.
10.Store at -80℃ for further use.
Protocol of QS
Preparation of bacteria solution
1. Culture engineered S. oneidensis MR-1（with PYYDT-GFP plasmid）and wild type S. oneidensis MR-1 MR-1(with empty PYYDT plasmid)
2. Measure the concentration of the bacteria by using spectrometer
3. Dilute the concentration of the bacteria at OD600=0.05（in 5ml YT medium with 50ng/ml Kan in the test tube）
4. Culture engineered S. oneidensis MR-1 MR-1（with PYYDT-GFP plasmid）and wild type S. oneidensis MR-1 MR-1(with empty PYYDT plasmid) until the medium starts to be turbid(when OD600 is around 0.1)
QS verification operation experiment
QS verification operation experiment
1. Add 200μl YT bacteria medium with 50ng/ml Kan, engineered S. oneidensis MR-1 MR-1（with PYYDT-GFP plasmid）and wild type S. oneidensis MR-1 MR-1(with empty PYYDT plasmid) solution in wells of 96×microplate
2. Measure the concentration and GFP intensity of each well by using Microplate Reader.
3. Shake the microplate for 20 minutes
4. Repeat step 2,3 for about 10 hours
5. Collect the data and then deal with it
Protocol of MO
Preparation of anaerobic mineral salt medium
1. Take 600 ml ddH2O
2. According to the required HEPES concentration (10~100mM)(11.91g/L for 50mM), add HEPES and stir to dissolve
3. After HEPES is dissolved, 100ml(10×) of macroelement reserve solution is added
4. Add 1ml(1000×)MgSO4·7H_2O concentrated solution
5. Add 10ml(100×) trace element reserve solution
6. Adjust PH to 7.0 to 7.2 with 4M KOH solution
7. Add sodium lactate (2.7885ml(60%)(20mM) to the electron donor
8. Use ddH_2O to a liter
9. Pack the medium into serum bottles, each 30ml
10. Treat each serum bottle with N_2 aeration for 15 minutes
11. Seal the serum bottles
12.Treat in autoclave for 20 minutes at 121℃
13. Sealed storage
Preparation of 100×M.O. solution
1 Add 50mg methyl orange into 1L absolute ethanol
2 Pack the solution into serum bottles
3 Treat each serum bottle with N_2 aeration for 15 minutes
4 Seal the serum bottles
5 Treat in autoclave for 20 minutes at 121℃
6 Sealed storage
Preparation of S. oneidensis MR-1
1 Measure the concentration of engineered S. oneidensis MR-1（with PYYDT plasmid）and wild type S. oneidensis MR-1 (with empty PYYDT plasmid)
2 Calculate the volume we need
3 Add the bacteria into 1.5ml EP tube with pipetting gun, then collect them by centrifugation.
4 Repeat the step3 until the concentration of bacteria in each EP tube is about 3(OD600)
5 Add 300μl prepared aerobic mineral salt medium into each EP tube with pipetting gun and then blow and mix the solution completely
MO operation process
1 Add 0.3ml 100×M.O. solution into anaerobic mineral salt medium with syringe
2 Add 0.03ml 1000×Kan(50ng/μl) solution into 1.5ml EP tube with pipetting gun
3 Move 0.03ml 1000×Kan(50ng/μl) solution from 1.5ml EP tube to anaerobic mineral salt medium with syringe, blow and mix them completely
4 Add 0.3ml prepared engineered S. oneidensis MR-1 MR-1（with PYYDT plasmid）and wild type S. oneidensis MR-1 MR-1 (with empty PYYDT plasmid) solution into anaerobic mineral salt medium with syringe (so we can control the concentration of bacterium is about 0.1(OD600) in each serum bottle
5 Move 200μl bacteria liquid from the each serum bottle into wells of the 96×Microplate
6 Measure the concentration of MO (measured under OD465) of each well
7 Incubate these samples at 30℃ for 20 minutes.
8 Repeat the step 5,6,7 until the OD465 of one group is lower than 0.1
9 Collect the data and deal with it
Standard MO operation process
1 Dilute the MO solution into concentration gradient at 0, 3, 6, 10, 20, 30, 40, 50(mg/L)
2 Add 200μl MO solution of each concentration gradient into wells of the 96×Microplate
3 Measure the concentration of MO (measured under OD465) of each well
4 Collect the data and deal with it
Protocal of aNAT
1.Preparation of aerobic mineral salt medium
2.Preparation of 2X YT medium
1 Add 16g Tryptone
2 Add 10g yeast extract
3 Add 10g NaCl
4 Dilute to 1L with ddH2O
5 Treat in autoclave for 20 minutes at 121℃
3.Pre-treatment of OD600 measurement
1. Cultivate bacteria in 2X YT medium for 12-16h at 30℃.
2. Measure OD600 of the bacteria suspension, then calculate the volume in proportion to absorbance of 0.05 when it is diluted to 5mL.
3. Centrifuge the volume of bacteria suspension calculated above at 4000 rpm for 3 minutes and we will see sediment in the tubes.
4. Remove the YT medium in the tubes, add some mineral salt medium, and resuspend.
5. Dilute the resuspension to 5mL with mineral salt medium. Add kanamycin to 50ug/mL.
6. Cultivate the dilution until it is a little bit turbid. (approximately 3-5h)
7. Add aromatic amine of different concentration to the dilution, then append the mix to 96-well plate.
4. Measurement condition settings on CLARIOstar
1. Mode: Absorbance, wavelength: 600nm
2. Endpoint mode, 52 cycles, 20 min/cycle, shake between each cycle at 200 rpm.
3. Layout:
| Blank | Blank | Blank |
| controlling | controlling | sample |
| controlling | controlling | sample |
| controlling | controlling | sample |
| controlling | controlling | sample |
| controlling | controlling | sample |
| controlling | controlling | sample |
| controlling | controlling | sample |