Team:Worldshaper-XSHS/Protocol
Protocols and methods
Plasmid DNA extraction
(1)Transfer 1.5mL of the culture to a Eppendorf tube. Centrifuge at 8000rpm for 5 minutes. Discard the supernatant. Add 280μL of resuspension solution(P1 buffer). Completely resuspent cell pellet.
(2)Add 200μL of lysis solution(Buffer P2) and mix gently. Set aside for 1 minute.
(3)Add 200 μL of neutralizing solution(P3 buffer) and mix by inverting the tubes for 5-10 times.
(4)Centrifuge at 12000rpm for 10 min and carefully transfer the supernatant to a adsorption column. Centrifuge at 8000rpm for 30sec, and remove the supernatant.Add 500μL of DW1 Buffer and centrifuge at 8000rpm for 30sec.
(5)Add 500μL of Wash Solution and centrifuge at 9000rpm for 30sec. Repeat twice.
(6)Centrifuge for 1min and open the lid for 1min to make the alcohol evaporate completely. Move the adsorption column to a new Eppendorf tube and add 30 μL of TE solution to obtain the plasmid DNA.
Transformation
(1) Thaw 100µL competent E. coli DH5α cells on ice until it is no longer frozen.
(2)Add 10µL ligation product into 100µL competent E. coli DH5α cells suspension, and then carefully flick the tube 4-5 times to mix cells and DNA.
(3)Place the mixture on ice for 30 minutes.
(4)Heat shock at exactly 42°C for exactly 90 seconds. Then place the mixture on ice for 1 minutes.
(5)Pipette 1 ml of room temperature LB media into the mixture. Incubate the mixture at 37°C with vigorous shaking for 60 minutes.
(6)Plate the mixture onto LB agar media containing antibiotic (ampicillin/ chloramphenicol). Incubate overnight at 37°C with plates upside down.
Preparing competent cells
Before we start: preparing freshly grown E. coli DH5α cells
(1)Preparing the CaCl2 solution at a concentration of 0.1mol/L
(2)Incubate 50µL E. coli DH5α cell suspension into 4ml LB media at 37°C with shaking in centrifugal speed of 200rpm overnight (without antibiotic).
(3)The next day, incubate the 1mL Bacterial suspension at 37°C with shaking in centrifugal speed of 200rpm until the OD600 is approximately 0,5(+-0,1)
(4)Add 25mL above incubated suspension into centrifuge (with gauge of 50mL) tube and centrifuge for 10min at 4 ℃ in centrifugal speed of 4000rpm.
(5)Discard the filtrate.
(6)Add 21ml of the prepared CaCl2 solution into precipitated bacteria. Incubate the mixture on ice for 20min.
(7)Centrifuge the above suspension for 10min at 4 ℃ in centrifugal speed of 4000rpm.
(8)Discard the filtrate.
(9)Add 3.4ml of the prepared CaCl2 solution (precooled) and 0.6mL of pure glycerol into the suspension.
(10)Split charge the product into eppendorf tubes, each 100µL, and cryopreservation at -80℃.
Gel Extraction
Make the plasmid through Enzyme digestion do the electrophoresis. Cut the pieces of agar under ultraviolet light. Use a gel extraction kit to extract target sequence from the gel just cut off:
(1)Tranfer the sample into a clean 1.5mL microcentrifuge tube.Add 1 volume Binding Buffer.
(2)Incubate at 50~60℃ for 7 minutes or until the gel has completely melted. Vortex or shake the tube every 2-3 minutes.
(3)Insert a HiBind DNA Mini Column in a 2mL Collection Tube.
(4)Add no more than 700uL DNA/agarose solution form step (2) to the HiBind DNA Mini Column.
(5)Centrifuge at 10,000g for 1 minute at room temperature.
(6)Discard the filtrate and reuse collection tube.
(7)Repaeat Steps (4)-(6) until the sample has been transferred to the column.
(8)Add 300uL Binding Buffer.
(9)Centrifuge maximum speed (≥13,000g) for 1minute at room temperature.
(10)Discard the filtrate and reuse collection tube.
(11)Add 700uL SPW Wash Buffer.
(12)Centrifuge at maximum speed for 1 minute at room temperature.
(13)Discard the filtrate and reuse collection tube.
(14)Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
(15)Transfer the HiBind DNA Mini Column to a clean 1.5mL microcentrifuge tube.
(16)Add 15-3uL Election Buffer or deionized water directly to the center of the column membrane.
(17)Let sit at room temperature for 2 minutes.
(18)Centrifuge at maximum speed for 1 minute at room temperature.
(19)Store DNA at -2℃.
Cycle-pure kit
(1)Transfer the sample into a clean 1.5mL microcentrifuge tube.
(2)Add 4-5volumes CP Buffer. For PCR products small than 200bp,add 6 volumes CP Buffer.
(3)Vortex to mix throughly. Briefly centrifuge to collect any drops from the inside of the lid.
(4)Insert a HiBind DNA Mini Column into a 2mL Collection Tube.
(5)Add the sample from step(3) to the HiBind DNA Mini Column.
(6)Centrifuge at maximum speed (≥13,000g)for 1 minute at room temperature.
(7)Discard the filtrate and reuse collection tube.
(8)Add 700uL DNA Wash Buffer.
(9)Centrifuge at maximum speed for 1 minute.
(10)Discard the filtrate and reuse collection tube.
(11)Repeat Steps 10-12 for a second DNA Wash Buffer step.
(12)Centrifuge the empty HiBind DNA Mini Column at maximum speed for 2 minutes to dry the column.
(13)Transfer the HiBind DNA Mini Column into a clean 1.5mL microcentrifuge tube.
(14)Add 30-50uL Elution Buffer,or sterile deionized water directly to the center of column matrix.
(15)Let sit at temperature for 2 minute.
(16)Centrifuge at maximum speed for 1 minute.
(17)Store DNA at -20℃.
COTA / sclac function test
In this experiment, the test dye is reactive red x (RR).
(1)Activate glycerin bacteria: take 1ml of LB liquid medium without antibiotics, add 20 μl of glycerin bacteria (which has been transferred into plasmid containing CotA or ScLac), and culture at 37 ℃ and 220 rpm for 5 hours.
(2)Take 50 μl of activated bacterial bacterial fluid in 40 ml kanamycin resistant LB medium overnight until OD600 = 0.6.
(3)The final concentration of 0.2 mM IPTG was added to the induced expression group （IPTG+）overnight.The other group was not added.
(4)The bacterial solution was centrifuged at 4000 rpm 4 ℃ for 10 min, then resuspended with 5 ml kanamycin resistant LB liquid medium, and centrifuged at 4000 rpm 4 ℃ for 10 min, repeated another time; 30ml of LB liquid medium containing kanamycin resistant was resuspended.
(5)30 μl Triton X-100 (the final concentration of Triton X-100 is 0.1% v / V) was added to 30 ml LB liquid medium containing kanamycin.
(6)Shaking at E.coli cells at 30 ℃ and 220 rpm for 30 min to increase their permeability.
(7)Centrifugation at 4000 rpm 4 ℃ for 10 min, suspension with 5 ml kanamycin resistant LB liquid medium, centrifugation at 4000 rpm 4 ℃ for 10 min then repeated another time.
(8)25 ml LB liquid medium containing kanamycin resistance was used to resuspend the bacteria.
In IPTG + group, 2.5ml 2mM RR and 5 μl 1M IPTG were added to 25ml CotA / ScLac bacterial solution.
In IPTG - group: 25 ml of CotA / ScLac was added with 2.5 ml of 2 mM RR.
(9)Each group was divided into 1ml centrifuge tubes and 2ml centrifuge tubes. The supernatant was centrifuged at 37 ℃, 150 rpm for 0 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 12000 rpm for 1 min and then centrifuged to determine 530nm absorbance.
Azr / Azor function test
Because the catalytic effect of AzR/ AzoR needs anaerobic environment, the bacterial solution was separated into 1.5ml EP tubes and then cultured in anaerobic condition.
(1)Overnight activated AzoR was expanded culturing until the OD600 = 0.6, and IPTG with final concentration of 0.2mM was added to induce to overnight culturing.
(2) After triton-100 cells were transfused in the same way as laccase, centrifugation was carried out at 4000 rpm for 10 min, suspension was carried out with 5 ml kanamycin resistant LB liquid medium, centrifugation was carried out at 4000 rpm for 10 min and repeated once.
(3)Use 20 ml LB liquid medium containing kanamycin resistance to suspend the bacteria again.
In the IPTG-CuCI2 + group, 20ml of AzR / AzoR bacterial solution was added with 25mM of CuCl2, 2ml of 2mM RR and 4 μl of 1M IPTG.
In the IPTG-CuCI2 group-, 2ml 2mM RR was added to 20ml of AzR / AzoR bacterial solution.
(4)Each tube was separately filled with 1.5ml centrifuge tube (about 1800 μ L), and cultured at 37 ℃, 150rpm for 0h, 4h, 8h, 12h, 24h, 48h, 12000rpm for 1min, then the supernatant was centrifuged to determine 530nm absorbance.
Wastewater degradation experiment
To prepare wastewater reactive red x (WR):
(1) Centrifugate the wastewater from the printing and dyeing plant at 4000 rpm for 10min and take the supernatant;
(2) Adjust the pH value. Take 18ML of supernatant, add 2ml of 20 × PBS buffer solution, prepare 10x active brilliant red mother liquor WR, with a concentration of 20mM.
Test on the ability of CotA / sclac to degrade dyes in wastewater:
(1) Activated glycerol bacteria. Take 20 μl (BL21 (DE3) strain containing plasmid CotA / ScLac) of target glycerin bacteria, add 1ml of LB liquid medium without antibiotic, and culture at 37 ℃ and 220 rpm for activation overnight;
(2)800 μl of activated bacterial solution was cultured in 30 ml kanamycin resistant LB medium for 8 hours, OD600 = 0.6.
(3)IPTG promoter protein was added to induce overnight, the final concentration of IPTG was 0.2mm.
(4)The induced bacterial solution was centrifuged at 4000 rpm and 4 ℃ for 10 min, then the supernatant was removed and resuspended with 5 ml kanamycin resistant LB liquid medium.
(5)Repeat step 4.
(6)Suspension with 50 ml LB liquid medium containing kanamycin;
(7)30 μl Triton X-100 (the final concentration of Triton X-100 is 0.1% v / V) was added to 30 ml LB liquid medium containing kanamycin. shaking the bacteria at 30 ℃ and 220 rpm permeabilize for 30 min.
(8)Centrifugation at 4000 rpm 4 ℃ for 10 min, suspension with 5 ml kanamycin resistant LB liquid medium, centrifugation at 4000 rpm 4 ℃ for 10 min, repeated once;
(9)The cells were resuspended with 27 ml kanamycin resistant LB solution. 6 μ l IPTG and 3 ml wastewater treatment solution WR 37 ℃ 150 rpm were added for 0 h, 24 h, 48 h and 72 h respectively.
EXPERIMENTAL SYSTEM
Table1 Double Digestion
|
|
Assemble digest |
Test digest |
|
|
50µL |
100µL |
10µL |
|
|
10×buffer |
5µL |
10µL |
1µL |
|
Enzyme I |
1µL |
2µL |
0.2µL |
|
Enzyme II |
1µL |
2µL |
0.2µL |
|
Plasmid |
1µg |
2µg |
200 ng |
|
ddH2O |
up to 50µL |
up to 100µL |
up to 10µL |
Table 2 Ligation
|
Materials |
Dosage |
|
10×T4 DNA Ligase Buffer |
2µL |
|
Vector DNA |
50 ng |
|
Insert DNA |
3×nvector DNA |
|
ddH2O |
up to 20µL |
|
T4 DNA Ligase |
1µL |
Table 3 Preparing LB media（100mL）
|
Materials |
Dosage of LB agar media |
Dosage of LB media |
|
Yeast-eatract |
0.5g |
0.5g |
|
Poptone-Y |
1g |
1g |
|
NaCl |
1g |
1g |
|
Agar |
1.5g |
— |
Table 4 DNA gel electrophoresis
|
Concentration |
Type |
Agarose H |
0.5×TBE |
EB |
|
1% |
Test |
20g |
20mL |
1µL |
|
Purification |
24g |
24mL |
1µL |
|
|
2% |
Test |
40g |
20mL |
1µL |
|
Purification |
48g |
24mL |
1µL |