Difference between revisions of "Team:Jilin China/Protocol"

1.PCR
From plasmid DNA template
Materials:
TransTaq○R HiFi DNA Polymerase
10×TransTaq HiFi Buffer II
2.5mM dNTPs
6×DNA Loading Buffer
10 μM forward Primer
10 μM reverse Primer
Plasmid DNA
ddH2O
PCR tube

Methods:
For a 50μL reaction:

Component	Volume	Final Concentration
Template Forward Primer (10 μM) Reverse Primer (10 μM) 10×TransTaq○R HiFi Buffer II 2.5 mM dNTPs TransTaq○R HiFi DNA Polymerase ddH2O	Variable(usually 50 ng) 1 μL 1 μL 5 μL 4 μL 0.5 μL Add to 50 μL	As required 0.2 μM 0.2 μM 1× 0.2 mM 2.5 units -
Total volume	50 μL	-

Combine all the component together into a PCR tube. Spin down briefly to collect the liquid to the bottom of the PCR tube. Put the PCR tube into a PCR machine.

NOTE: The TransTaq○R HiFi DNA Polymerase should be added last.

Settings of PCR machine:

Step	Temperature(℃)	Time
Initial denaturation	94	5 min
35 cycles	94 50-60 72	30 sec 30 sec 1-2 kb/min
Final extension	72	5 min
Hold	4	∞

2.Agarose gel electrophoresis
Materials:
Agarose powder
TAE buffer
Gel mould
Gel comb
Gel Tank
Ethidium bromide
DNA marker DL10000
6× DNA loading dye
Methods:
1.Prepare 1% w/v solution agarose powder in 1×TAE buffer(e.g. 1g agarose powder in 100mL 1×TAE buffer).
2.Heat the mixture in microwave oven until agarose is completely dissolved.
3.Cooling the solution till around 50℃ , add Ethidium bromide to 0.1‰ v/v final concentration.
4.Pour the solution into a gel mould.
5.Put the solution at room temperature till the gel is set.
6.Remove the comb and transfer the agarose gel to a gel tank.
7.Mix DNA samples with proper amount of 6× DNA loading buffer and load the samples and DNA marker into the gel.
8.Run the gel for 30 minutes of 5V/cm.

3.Gel extraction
Gel extraction was performed according to the TIANgel Midi Purification Kit

4.Digestion
Materials:
Restriction Enzyme (NEB)
10× cutsmart buffer (NEB)
Plasmid DNA or DNA fragment
ddH2O
Methods:

Component	Test digestion (double digestion)	Assemble digestion(double digestion)
Restriction enzyme 10× cutsmart buffer Plasmid DNA or DNA fragment ddH2O	0.5 μL + 0.5 μL 1 μL Variable (~ 500 ng) Add to 10 μL	3 μL + 3 μL 5 μL Variable (~ 3 μg) Add to 50 μL

5.DNA purification
PCR purification was performed according to the TIANquick Midi Purification Kit

6.Ligation
T4 DNA Ligase
10× T4 DNA ligase buffer
Vector DNA
Insert DNA
Microcentrifuge tube
ddH2O
Methods:

Component	Volume
Vector DNA Insert DNA 10× T4 DNA ligase buffer T4 DNA Ligase ddH2O	Variable (~50 ng) Variable (molar ratio of vector:insert=1:3) 2 μL 1 μL Add to 20 μL

1.Add all the components list in the table above to a microcentrifuge tube.
2.Incubate the ligation mixture at 16℃ over night.

7.Chemical Transformation
Materials:
Ice
SOC medium
Selection plates
Chemically competent cell
Plasmid DNA or ligation reation mix
Methods: 1. Put 50 μL chemical competent cells on ice for 10 minutes.
2. Add 100ng DNA of known plasmid or 10 μL ligation reaction mix into the competent cells .
3. Softly flick the tube 4-5 times to mix cells and DNA.
4. Put the tube on ice for 30 minutes.
5. Heat shock at exactly 42℃ for exactly 45 seconds.
6. Put the tube on ice for 2 minutes.
7. Add 950 μL SOC medium into the tube.
8. Incubate at 37℃ ,200rpm for 60 minutes.
9. Spread:
   For known plasmid:
     Transfer 100 μL of transformation cell mixture onto a selection plate.
   For ligation reaction mix:
     Centrifuge the whole 1000 μL transformation cell mixture at 6000rpm for 2 minutes.
     Remove 900 μL supernatant and re-suspend cells with 100 μL supernatant left. Transfer the 100 μL resuspended cells onto a selection plate.
10. Incubate overnight at 37℃ with plates upside down.

8.Plasmid DNA extraction
Plasmid DNA extraction was carried out according to the TIANprep Rapid Mini Plasmid Kit
EGFP Measurement
Materials:
Liquid LB medium
100mg/mL Kanamycin
BL21 competent cells
Plasmids: pET28a vector, pET28a-J23114-Dmpr-GFP-Rluc, pET28a-J23101-Dmpr-GFP-Rluc, pET28a-J23107-Dmpr-GFP-Rluc, pET28a-Pr-Dmpr-GFP-Rluc, pET28a-Pr-PEKDmpr-GFP-Rluc,
Methods:
1. Transform plasmids into BL21 competent cells. Incubate the plates at 37°C overnight.
3. Pick a single colony and inoculate into liqud LB medium containing kanamycin. Overnight culture.
4.Seal the tubes and incubate overnight at 37°C shaking at 200-250 rpm

4.Bacterial Glycerol Stocks:
Materials:
0.2mL sterile 40% glycerol solution
0.2mL bacteria liquid
Methods:
1.Put 0.2ml bacterial culture in a sterile eppendorf tube
2.Add 0.2ml sterile 80% glycerol solution and mix well
3.Seal the eppendorf tube and store at -80°C

5.Bacterial amplification:
Materials:
15mL LB medium
7.5 L 100mg/mL Kanamycin
50mL centrifugal tubes
Methods:
1.Add 15 ml liquid LB medium into 50mL centrifugal tubes
2.Add 7.5 L 100mg/mL Kanamycin into the medium
3.Add 1mL bacteria liquid
4.Seal the centrifugal tubes and incubate at 37°C shaking at 200-250 rpm

6.Detection of EGFP:
Materials:
96-well plates
Methods:
1.Take 100 L samples of the 50mL centrifugal tubes into the 96-well plates
2.Use Microplate reader to shake the 96-well plates and measure the samples (OD600 and EGFP) at 0, 2, 4, 6, 8, 10, 12 and 24 hours of amplification
Renilla Luciferase Assay
Materials:
60 mM Promega ViviRen Renila Luciferase substrate
1 M Phenol solution
1 M 2-Chlorophenol solution
1 M 4-Chlorophenol solution
1 M 2,4-Dichlorophenol solution
1 M Catechol solution
Methods:
1.Take 595μlbacteria liquid from 15mL culture tube and add into 1.5mL eppendorf tube
2.Mix 6 L Phenols into 595 L bacteria liquid in the 1.5mL eppendorf tube
3.For J23114, J23101, J23107: Seal the centrifugal tubes and incubate at 37°C shaking at 200-250 rpm for 3h
4.For wt-pr, wt-prpk: Seal the centrifugal tubes and incubate at 37°C shaking at 200-250 rpm for 12h
5.Take 45 L mixture from 1.5mL eppendorf tube and add into new 1.5mL eppendorf tube
6.Mix 5 L 0.6mmol/mL Luciferase substrate Renila into the mixture in 1.5mL eppendorf tube
7.Vortex 7 seconds and detect the Luciferase

1.Detoxification curve
materials：
arabinose;
IPTG
LB culture medium (with 50μg/mL kanamycin;)
Microplate reader
Methods：
1.Inoculated 7.5 mL overnight cultured bacteria into 800mL LB media added kanamycin, making its initial value of Abs600 around 0.015.
2.When value of Abs600 reached 0.15-0.2, sub-packed 150 mL medium into a conical flask. Add 0.2% arabinose into the rest 650mL medium.
3.When OD600 was steady, added IPTG(0.1mM) into the medium.
4.Abs600 measurement began at inoculation, every 1h in the beginning 6hours and every 2hours in the next 6hours, respectively.

2.poisoning curve
materials：
arabinose;
LB culture medium (50μg/ml kanamycin;)
Microplate reader
Methods：
Inoculated 7.5 mL overnight cultured bacteria into 800mL LB media added kanamycin, making its initial value of Abs600 around 0.015. measured Abs600 every hour. When value of Abs600 reached 0.15-0.2, sub-packed medium into 4 conical flasks, 150mL each. Then added 0%, 0.1%, 0.2%, 0.6% arabinose respectively, measured Abs600 every 1h in the beginning 6hours and every 2hours in the next 6hours, respectively.

3.IPTG curve
materials：
IPTG
LB culture medium(kanamycin;)
Microplate reader
Methods：
1.the bacterio-liquid(7.5ml)shaken overnight at 37℃ were transferred into LB culture medium(800ml) with kanamycin(1b).
2.Measure its abs600 once an hour when abs600 reaches 0.015.
3.Sub-pack in four Conical flasks with 150ml adding different concentrations of IPTG（0.1mM 0.2mM 0.5mM），when abs600 reaches 0.150-0.200.
4.Since transferring ,measure abs600 once an hour in the first six hour ,and then once two hour.

Inoculated 7.5 mL overnight cultured bacteria into 800mL LB media added kanamycin, making its initial value of Abs600 around 0.015. measured Abs600 every hour. When value of Abs600 reached 0.15-0.2, sub-packed medium into 4 conical flasks, 150mL each. Then added 0.1mM, 0.2mM, 0.5mM IPTG respectively, measured Abs600 every 1h in the beginning 6hours and every 2hours in the next 6hours, respectively.

1.Phenol degradation experiments
Materials and methods
2%(m/v) 4-amino anti pyrine (4-AAP), 8%(m/v) potassium ferricyanide K3Fe(CN)6, 1mM glucose, 0.9% NaCl, buffer solution: 10g NH4Cl in 50mL ammonium hydroxide.
Phenol can react with 4-AAP in alkaline medium (pH =10.0±0.2), with the oxidizer K3Fe(CN)6. Reaction product antipyrine dye appears orange, whose absorption peak is at 510nm.

4-AAP assay
For a 14.5ml reaction system:

Component	Volume
samples H2O buffer 2% 4-AAP 8% K3Fe(CN)6	0.5ml 9.5 ml 0.9 ml 1.8ml 1.8ml
Total volume	14.5ml

Experimental group:
1mM phenol, 1mM glucose, 0.9%NaCI solution with tfdB-JLU bacteria 0h
2mM phenol, 1mM glucose, 0.9%NaCI solution with tfdB-JLU bacteria 0h
5mM phenol, 1mM glucose, 0.9%NaCI solution with tfdB-JLU bacteria 0h
1mM phenol, 1mM glucose, 0.9%NaCI solution with tfdB-JLU bacteria 20h
2mM phenol, 1mM glucose, 0.9%NaCI solution with tfdB-JLU bacteria 20h
5mM phenol, 1mM glucose, 0.9%NaCI solution with tfdB-JLU bacteria 20h
Control group:
2mM phenol, 1mM glucose, 0.9%NaCI solution with empty vector bacteria 20h
Blank:
0mM phenol, 1mM glucose, 0.9%NaCI solution with supernatant of tfdB-JLU bacteria

Phenol disposing system with tfdB-JLU bacteria
1. E. coli harboring the cloned pET-28a-tfdB-JLU was incubated in 3 mL LB media supplemented with 30μg/mL of kanamycin at 37℃ on a rotary shaker operating at 220 rpm at 37℃ overnight.
2. Enlarged overnight cells. The cells were grown until the value of OD600 reached 0.4. Cooled down the medium untill it reached 16℃, 0.2mM IPTG was then added, and the cells were incubated in 16℃ for 12h by shaking at 220rpm to induce tfdB-JLU enzyme.
3. Took out 20ml inducted bacteria into 50mL concentrator bowls, marked 1, 2, 3, then centrifuged in 3000rpm for 5min, discarded supernatant, resuspended with 10mL 1mM glucose and 0.9% NaCl solution, centrifuged in 3000rpm for 5min, took out 1mL supernatant into 1ml EP tube, marked 0-0. Discarded concentrator bowls’ supernatant, then resuspended with 10mL 1, 2, 5mM phenol with 1mM glucose, 0.9% NaCl solution and centrifuged in 3000rpm for 5min. took out 1mL supernatant marked 1-0, 2-0, 3-0. Resuspended again and took 5mL sample for measuring OD600. Put concentrator bowls in 25℃ for further disposing.
Phenol disposing system with empty vector bacteria
1. E. coli harboring the cloned pET-28a was incubated in 3 mL LB media supplemented with 30μg/mL of kanamycin at 37℃ on a rotary shaker operating at 220 rpm at 37℃ overnight.
2. Enlarged overnight cells. The cells were grown until the value of OD600 reached 0.4. Cooled down the medium till it reached 16℃, then incubated for 12h by shaking at 220rpm.
3. Took out 20ml inducted bacteria into 50mL concentrator bowls, marked empty vector, then centrifuged in 3000rpm for 5min, discarded supernatant, resuspended with 10mL 1mM glucose and 0.9% NaCl solution, centrifuged in 3000rpm for 5min. Discarded concentrator bowls’ supernatant, then resuspended with 10mL 2mM phenol with 1mM glucose, 0.9% NaCl solution and centrifuged in 3000rpm for 5min. took out 1mL supernatant marked empty vector-0. Resuspended again and put concentrator bowls in 25℃ for further disposing.
4. Centrifuged empty vector-0 in 12000rpm for 5min, took out 0.5mL sample into 10mL concentrator bowl for assay system. Incubated 10min and measured A510. (blanked with water)
5. The high-performance liquid chromatography (HPLC) column was calibrated initially with the mobile phase (methanol and 0.2% acetic acid 30:70) at a flow rate of 1 mL/min for 20 min. The detection wavelength was at 275 nm.
6. Prepared 1mM, 2mM, 5mM phenol PBS solution and 1mM, 2mM catethol PBS solution. Measured with HPLC and recorded retention time and peak area.
7. Day 0: inoculated tfdB-JLU and empty vector bacteria strain.
8. Day 1: enlarged overnight bacteria, cells were grown until the value of OD600 reached 0.4. 0.3mM (IPTG) was then added, and the cells were incubated in 18℃ for 16-24h by shaking at 220rpm for induction.
9. Took 1mL tfdb and 1mL empty vector bacteria into 1mL EP tube, centrifuged in 4000rpm for 10min, discarded supernatant, resuspended with 1mM, 2mM, 5mM phenol respectively, incubated for 1h, 2h, 4h, 20h, then measured with HPLC.
10.
11. Experimental group
12. 1mM phenol disposed with tfdB-JLU bacteria for 0h
13. 2mM phenol disposed with tfdB-JLU bacteria for 0h
14. 5mM phenol disposed with tfdB-JLU bacteria for 0h
15. 1mM phenol disposed with tfdB-JLU bacteria for 1h
16. 2mM phenol disposed with tfdB-JLU bacteria for 1h
17. 5mM phenol disposed with tfdB-JLU bacteria for 1h
18. 1mM phenol disposed with tfdB-JLU bacteria for 2h
19. 2mM phenol disposed with tfdB-JLU bacteria for 2h
20. 5mM phenol disposed with tfdB-JLU bacteria for 2h
21. 1mM phenol disposed with tfdB-JLU bacteria for 4h
22. 2mM phenol disposed with tfdB-JLU bacteria for 4h
23. 5mM phenol disposed with tfdB-JLU bacteria for 4h
24. 1mM phenol disposed with tfdB-JLU bacteria for 20h
25. 2mM phenol disposed with tfdB-JLU bacteria for 20h
26. 5mM phenol disposed with tfdB-JLU bacteria for 20h
27.
28. Control group:
29. 1mM phenol disposed with empty vector bacteria for 0h
30. 2mM phenol disposed with empty vector bacteria for 0h
31. 5mM phenol disposed with empty vector bacteria for 0h
32. 1mM phenol disposed with empty vector bacteria for 1h
33. 2mM phenol disposed with empty vector bacteria for 1h
34. 5mM phenol disposed with empty vector bacteria for 1h
35. 1mM phenol disposed with empty vector bacteria for 2h
36. 2mM phenol disposed with empty vector bacteria for 2h
37. 5mM phenol disposed with empty vector bacteria for 2h
38. 1mM phenol disposed with empty vector bacteria for 4h
39. 2mM phenol disposed with empty vector bacteria for 4h
40. 5mM phenol disposed with empty vector bacteria for 4h
41. 1mM phenol disposed with empty vector bacteria for 20h
42. 2mM phenol disposed with empty vector bacteria for 20h
43. 5mM phenol disposed with empty vector bacteria fo

Overexpression and purification of target enzyme
Day 1
E. coli harboring the cloned pET-28a was incubated in LB media supplemented with 50μg/mL of kanamycin at 37℃ on a rotary shaker operating at 220 rpm at 37℃ overnight.
Day2
Suspend overnight cells. The cells were grown until the value of OD600 reached 0.4. 0.3mM (IPTG) was then added, and the cells were incubated in 18℃ for 16h by shaking at 180 rpm to induce tfdB-JLU enzyme.
Day3
Harvest overnight-inducted cells. The harvested cells were centrifuged at 4000rpm for 30 min in 4℃, and supernatant was then discarded. The cells were resuspended in 50 mL of lysis buffer (200mM NaCl, 20% glycerol, 20mM Tris–HCl, and pH 7.5) and sonicated 5s with 5s interval each time at 20 kHz for 1h on ice. The cell debris was removed by centrifugation at 12000rpm for 30 min. The His-tagged recombinant enzyme was purified by passing the supernatant through a Ni-NTA His Bind column. The column was washed with wash buffer (20 mM Tris-HCl buffer, pH 7.5, containing 20% (v/ v) glycerol, 200mM NaCl and 20mM, 50mM, 100mM imidazole grads) and eluted with elution buffer ((20 mM Tris-HCl buffer, pH 7.5, containing 20% (v/ v) glycerol, 200mM NaCl and 250mM imidazole).
Enzyme assay and characterisation
The activities of chlorophenol hydroxylases were determined by monitoring the decrease in absorbance at 340 nm (e340 = 6,220 M-1 cm-1) following the substrate-dependent oxidation of NADPH. Unless otherwise indicated, standard enzyme activity assays were performed by incubating the purified enzyme with 0.1 mM 2,4-DCP and 0.2 mM NADPH in 50 mM sodium phosphate buffer (pH 7.5) at 25℃ in 1 ml. One unit of activity was defined as the amount of enzyme required to consume 1 μmol NADPH per min at 25℃.