Team:Jilin China/Protocol
1. PCR
From plasmid DNA template
Materials:
TransTaq® HiFi DNA Polymerase
10×TransTaq® HiFi Buffer II
2.5 mM dNTPs
6× DNA Loading Buffer
10 μM Forward Primer
10 μM Reverse Primer
Plasmid DNA
ddH2O
PCR tube
Methods:
For a 50 μL reaction:
| Component | Volume | Final Concentration |
|---|---|---|
|
Template Forward Primer (10 μM) Reverse Primer (10 μM) 10×TransTaq® HiFi Buffer II 2.5 mM dNTPs TransTaq® 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 components 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® HiFi DNA Polymerase should be added last.
Settings of PCR machine:
| Step | Temperature(℃) | Time |
|---|---|---|
| Initial denaturation | 94 | 5 mins |
| 35 cycles | 94 50-60 72 |
30 sec 30 sec 1-2 kb/min |
| Final extension | 72 | 5 mins |
| 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 agarose solution with 1×TAE buffer(e.g. 1 g agarose powder in 100 mL 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 amounts of 6× DNA loading buffer and load the samples and DNA marker into the gel.
8.Run the gel for 30 minutes of 5 V/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 |
1.Mix all the components following the table above.
2.Incubate the digestion mixture at 37℃ for 1~2 hrs.
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 listed in the table above to a microcentrifuge tube.
2.Incubate the ligation mixture at 16℃ overnight.
7. Chemical Transformation
Materials:
Ice
SOC medium
Selection plates
Chemical competent cell
Plasmid DNA or ligation reation mix
Methods:
1. Put 50 μL chemical competent cells on ice for 10 minutes.
2. Add 100 ng 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 mins.
5. Heat shock at exactly 42℃ for exactly 45 secs.
6. Put the tube on ice for 2 mins.
7. Add 950 μL SOC medium into the tube.
8. Incubate at 37℃ ,200 rpm for 60 mins.
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 6000 rpm for 2 mins.
Remove 900 μL supernatant and re-suspended 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
Materials:
Liquid LB medium
100 mg/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,
transparent flat bottom 96-well plates (for Absorbance 600 measurement),
white flat bottom 96-well plates (for EGFP measurement)
Methods:
1. Transform plasmids into BL21 competent cells. Incubate the plates at 37°C overnight.
2. Pick one monoclonal colony and inoculate into 5 mL liqud LB medium containing 50 μg/mL kanamycin. Overnight culture.
3. Transfer 1 mL overnight-cultured bacteria into 15 mL LB medium containing 50 μg/mL kanamycin.
4. Take 100 μL samples from the 15 mL growing bacteria at 0, 2, 4, 6, 8, 12 and 24 hours and transfer into transparent flat bottom 96-well plates for Absorbance 600 measurement or white flat bottom 96-well plates for EGFP detection.
5. Measure Absorbance 600 or EGFP by Biotek microplate reader at indicated time.
Materials:
600 μM Promega ViviRen Renila Luciferase substrate
100 mM phenol solution
100 mM 2-chlorophenol solution
100 mM 4-chlorophenol solution
100 mM 2, 4-dichlorophenol solution
100 mM pyrocatechol solution
Liquid LB medium
100 mg/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.
2. Pick one monoclonal colony and inoculate into 50 mL LB medium containing 50 μg/mL kanamycin. Overnight culture.
3. Transfer 594 μL of 50 mL overnight bacteria into 1.5 mL eppendorf tubes.
4. Add 6 μL phenols into the aliquot 594 μL bacteria in the eppendorf tubes.
5. Seal the tubes and incubate at 37°C, 250 rpm, 3 hrs for vector, J23114, J23101 and J23107 groups. Incubate at 37°C, 250 rpm, 12 hrs for vector, Pr-DmpR and Pr-PEKDmpR groups.
6. Take 45 μL bacteria containing phenols mixture from tubes and put it into a new 1.5 mL eppendorf tube. Transfer corresponding 100 μL mixture to the transparent flat bottom 96-well plates as well.
7. Add 5 μL 600 μM renilla luciferase substrate into the 45 μL mixture in 1.5 mL eppendorf tube.
8. Vortex 7 seconds and measure the luciferase reading.
9. Measure the Absorbance 600 of the corresponding 100 μL mixture in the transparent flat 96-well plates.
Materials:
10% L-arabinose
LB medium
100 mg/mL Kanamycin
Methods
1. Inoculate 7.5 mL overnight cultured bacteria into 800 mL LB medium with 50 μg/mL kanamycin. Make sure the initial reading of Absorbance 600 is around 0.015.
2. Sub-pack the bacteria into 4 conical flasks, 150 mL each at Absorbance 600 around 0.15-0.2.
3. Add L-arabinose to the 4 conical flasks for 0%, 0.1%, 0.2%, 0.6% final concentration respectively.
4. Measure Absorbance 600 at once after inoculation. Every hour in the beginning 6 hrs and every 2 hrs in the next 6 hrs respectively.
Materials:
100 mM IPTG
LB medium
100 mg/mL Kanamycin
Methods
1. Inoculate 7.5 mL overnight cultured bacteria into 800 mL LB medium with 50 μg/mL kanamycin. Make sure the initial reading of Absorbance 600 is around 0.015.
2. Sub-pack the bacteria into 4 conical flasks, 150 mL each at Absorbance 600 around 0.15-0.2.
3. Add IPTG to the 4 conical flasks for 0, 0.1, 0.2, 0.5 mM final concentration respectively.
4. Measure Absorbance 600 at once after inoculation. Every hour in the beginning 6 hrs and every 2 hrs in the next 6 hrs respectively.
Materials:
10% L-arabinose
100 mM IPTG
LB medium
100 mg/mL Kanamycin
Plasmids: pET28a vector, T-A (pET28a-totion-antitoxin)
Methods:
1. Inoculate 7.5 mL overnight cultured bacteria into 800 mL LB medium with 50 μg/mL kanamycin. Make sure the initial reading of Absorbance 600 is around 0.015.
2. Sub-pack 150 mL bacteria into a new conical flask as the no IPTG and no L- arabinose control at Absorbance 600 around 0.15-0.2.
3. Add 13 ml L-arabinose into the rest 650 mL bacteria (0.2% final concentration).
4. Take 150 mL bacteria into a new conical flask as the no IPTG control when Absorbance 600 does not change any more.
5. Sub-pack 150 mL bacteria into a new conical flask and add 150 μl IPTG into the 150 mL bacteria (0.1 mM final concentration) when Absorbance 600 does not change any more.
6. Measure the Absorbance 600 at inoculation beginning. Every hour in the beginning 6 hrs and every two hours in the next 6 hrs respectively.
Materials
IPTG
LB medium
100 mg/ml Kanamycin
Ni-NTA His Bind column
lysis buffer: 200 mM NaCl, 20% glycerol, 20 mM Tris–HCl, and pH 7.5
buffer A: 20 mM Tris-HCl buffer, pH 7.5, containing 20% (v/v) glycerol, 200 mM NaCl
buffer B: 500 mM imidazole，20 mM Tris-HCl buffer, pH 7.5, containing 20% (v/ v) glycerol, 200 mM NaCl
Methods:
Day 1
1. Inoculate E. coli harboring tfdB-JLU in LB medium supplemented with 50 μg/mL kanamycin at 37℃, 220 rpm, overnight culture.
Day 2
2. Transfer 1 mL overnight cultured cells into 200 mL LB.
3. Add IPTG to final concentration 0.3 mM at OD600 around 0.4 of the grown bacteria.
4. Incubate the cells at 18℃ for 16 hrs by shaking at 180 rpm to induce tfdB-JLU enzyme.
Day 3
5. Harvest overnight-inducted cells. Centrifuge at 4000 rpm for 30 mins at 4℃. Discard the supernatant.
6. Resuspend with 50 mL lysis buffer (200 mM NaCl, 20% glycerol, 20 mM Tris–HCl, and pH 7.5).
7. Sonicate 5 s with 5 s interval each time at 20 kHz for 1 hr on ice.
8. Remove cell debris by centrifugation at 12000 rpm for 30 mins.
9. Purify the His-tagged recombinant enzyme by passing the supernatant through a Ni-NTA His Bind column.
10. Reload the supernatant twice before washing.
11. Use 8 mL volumes of buffer A wash the column.
12. Wash the column with 10 mL buffer A containing 20 mM imidazole (using buffer A dilute buffer B).
13. Collect samples, 1 mL/tube.
14. Wash the column with 10 mL buffer A containing 50 mM imidazole, collect samples, 1 mL/tube.
15. Wash the column with 6 mL buffer A containing 100 mM imidazole, collect samples, 1 mL/tube.
16. Wash the column with 10 mL buffer A containing 250 mM imidazole, collect samples, 1 mL/tube.
17. Wash the column with 40 mL buffer B.
18. Wash the column with 40 mL buffer A.
19. Wash the column with 40 mL H2O.
Materials:
nicotinamide adenine dinucleotide phosphate (NADPH)
FAD (flavin adenine dinucleotide)
sodium phosphate buffer (pH 7.5)
phenol substrate：2,4-DCP, 3-CP, 4-CP, phenol, 2,3-DCP, 2,5-DCP, 2,6-DCP, 3,4-DCP, 3,5-DCP, 2,4,5-TCP
Methods:
Determine the activities of chlorophenol hydroxylases by monitoring the decrease in absorbance at 340 nm (e340 = 6,220 M-1 cm-1) following the substrate-dependent oxidation of NADPH. Define one unit of activity as the amount of enzyme required to consume 1 μmol NADPH per min at 25℃.
1. Incubate 12 μg/mL tfdB-JLU with 0.1 mM substracts, 5 μM FAD and 0.2 mM NADPH in 50 mM sodium phosphate buffer (pH 7.5) at 25℃ in 1 mL mixture system.
2. Monitor Absorbance 340 for 1 hr.
Principles:
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.
Materials:
2% (m/v) 4-amino anti pyrine (4-AAP)
8% (m/v) potassium ferricyanide K3Fe(CN)6
1 mM glucose
0.9% NaCl
buffer: 10g NH4Cl in 50 mL ammonium hydroxide.
Methods:
1. Inoculate E. coli containing tfdB-JLU or mock in LB with 50 μg/mL kanamycin at 37℃, 220 rpm, overnight culture.
2. Transfer 1 mL overnight cultured cells into 200 mL LB.
3. Add IPTG to final concentration 0.3 mM at OD600 around 0.4 of the grown bacteria.
4. Incubate at 18℃ for 16 hrs by shaking at 180 rpm to induce tfdB-JLU enzyme.
5. Take 500 μL induced bacteria samples for measuring Absorbance 600.
6. Take out 20 mL corresponding bacteria into 50 mL centrifuge tube.
7. Centrifuge at 3000 rpm for 5 minw, discard supernatant, and suspend with 10 mL solution (1 mM glucose in 0.9% NaCl).
8. Centrifuge in 3000 rpm for 5 mins again. Discard supernatant.
9. Re-suspend with 10 mL 0, 1, 2, or 5 mM phenol with 1 mM glucose in 0.9% NaCl solution.
10. Incubate at 25℃ for 12 hrs.
11. Centrifuge samples immediately at 12000 rpm for 5 mins.
12. Take out 0.5 mL supernatant into assay system. Mix a 14.5 mL reaction system as below. Incubate for 10 mins.
| Component | Volume |
|---|---|
|
samples H2O buffer 2% 4-AAP 8% K3Fe(CN)6 |
0.5 mL 9.5 mL 0.9 mL 1.8 mL 1.8 mL |
| Total volume | 14.5 mL |
13. Measure Absorbance 510 for all the samples.
Materials:
ZORBAX Eclipse XDB C-18 column
methanol, acetic acid, phenol,
sodium phosphate buffer (pH 7.5)
LB medium with kanamycin
IPTG
HPLC condition:
ZORBAX Eclipse XDB C-18 column
mobile phase：methanol : 0.2% acetic acid (30:70)
rate: 1 mL/min
injection volume: 10 μL
wavelength: 275 nm
temperature: 25°C
Methods:
1. Inoculate E. coli containing tfdB-JLU or mock in LB medium with 50 μg/mL kanamycin at 37℃, 220 rpm, overnight culture.
2. Transfer 250 μL overnight cultured cells into 50 mL LB.
3. Add IPTG to final concentration 0.3 mM at OD600 around 0.4 of the grown bacteria.
4. Incubate at 18℃ for 16 hrs by shaking at 180 rpm to induce tfdB-JLU enzyme.
5. Take 1 mL induced bacteria samples for measuring Absorbance 600.
6. Transfer 1 mL corresponding induced bacteria samples to the tubes. Centrifuge 4000 rpm for 10 mins. Discard the supernatant.
7. Resuspend the precipitate with 0, 1, 2, or 5 mM phenol in PBS respectively. Incubate at 25℃, shaking for 4 hrs.
8. Centrifuge 4000 rpm for 10 mins. Keep the supernatant and pass the 0.22 μm filter.
9. Measure with HPLC.
Materials:
phenol
LB with kanamycin
HPLC assay materials and condition
4-AAP assay materials and condition
Methods:
1. Inoculate E. coli containing J23107-DmpR-tfdB-JLU or mock in LB with 50 μg/mL kanamycin at 37℃, 220 rpm, overnight culture.
2. Transfer 500 μL overnight cultured cells into 100 mL LB. Shake at 37 ℃ for 2 hrs.
3. Add 0, 1, 2, or 5 mM phenol into the cultured bacteria.
4. Measure Abs600 right after phenol adding and subsequent every 2 hrs till 24 hrs culture.
5. Phenol was tested 2, 6, 10 or 24 hrs after its adding with both HPLC method and 4-AAP assay.
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