Team:NCKU Tainan/Experiments
Colony PCR
Equipment:
1. PCR tubes
2. Ice
3. Thermocycler
4. Pipette and pipette tips
Consumables:
1. MQ or ddH2O
2. 10x PCR buffer
3. dNTPs 2.5 mM
4. Forward and reverse primer (10 μM)
5. Taq polymerase with 3 μl vent
1. On ice, add all components in a PCR tube, making up to 50 µl volume reaction.
| Components | Volume (μl) |
|---|---|
| MQ or ddH2O | 1.1 |
| 10x PCR buffer | 1 |
| dNTP 2.5 mM | 0.8 |
| Template | 1 |
| Forward primer 10 μM | 1 |
| Reverse primer 10 μM | 0.1 |
| Taq polymerase + vent | 0.1 |
| Total Volume | 10 |
2. Gently mix the PCR reactions and centrifuge briefly.
3. Transfer the PCR tubes to a thermocycler.
| Step | Temperature | Time |
|---|---|---|
| Initial denaturalization | 94°C | 5 mins |
| 25 - 35 cycles | 94°C (denaturation) | 30 secs |
| 55°C (annealing) | 30 secs | |
| 72°C (extension) | 2 mins (depend on sequence size 2 kbp/min.) | |
| Final extension | 72°C | 5 mins |
| Hold | 16°C (holding for a short time) or 4°C (holding for a long time) | ∞ |
Electroporation Escherichia coli for homologous
Equipment:
1. Pipette and pipette tips
2. Eppendorf tubes
3. Centrifuge
4. Flasks
5. Electroporation cuvettes (2 mm gap)
6. Electroporator
Consumables:
1. SOB
2. Chilled 10% glycerol
3. SOC
4. Selection plates
5. DNA fragment
6. 10% L-arabinose
7. Ampicillin
Competent cell making:
1. Inoculate 500 μl of overnight culture (Escherichia coli with pKD46) to 50 ml of fresh SOB-Mg medium with 50 μl of ampicillin at 30°C.
2. Grow cells with shaking at 30°C for 1 hour and 50 mins.
3. Add 1 ml of 10% L-arabinose and culture for 1 hour.
4. Transfer the culture into 50 ml centrifuge tube, and centrifuge it for 5 min at 4°C and 6,000 rpm to harvest cells. (After this stage, the cells should be kept cool throughout the cell preparation)
5. Remove the supernatant and suspend the cells with 1 ml of chilled 10% glycerol. Transfer the cell suspension into a 1.5 ml tube and centrifuge again for 1 min at 4°C and 12,000 rpm. (first wash)
6. Remove the supernatant using a pipette and suspend the cells with 1 ml of chilled 10% glycerol. Centrifuge again for 1 min at 4°C and 12,000 rpm. (second wash)
7. Remove the supernatant using a pipette and suspend the cells with 1 ml of chilled 10% glycerol. Centrifuge again for 1 min at 4°C and 12,000 rpm. (third wash)
8. Remove the supernatant using a pipette and suspend the cells with 250 μl (depends on the amount of the cell) of chilled 10% glycerol.
Electroporation:
1. Mix 50 μl of the competent cell and 5 μl DNA solution in a fresh 1.5 ml tube.
2. Transfer the mixture into chilled electroporation cuvette (2 mm gap), and pulse (Ec 2).
3. Add 1 ml of SOC with 20 μl 10% arabinose immediately after the pulse.
4. Incubate it for 1 hour at 37°C.
5. Plate 100 μl of the culture on a selection plate, and incubate at 30°C.
Electroporation Escherichia coli with plasmid
Equipment:
1. Pipette and pipette tips
2. Eppendorf tubes
3. Centrifuge
4. Flasks
5. Electroporation cuvettes (2 mm gap)
6. Eletroporator
Consumables:
1. SOB
2. Chilled 10% glycerol
3. SOC
4. Selection plates
5. DNA plasmid
Competent cell making:
1. Inoculate 1 ml of overnight culture to 50 ml of fresh SOB-Mg medium.
2. Grow cells with shaking at 37°C for 1.5 hours (recA+ strain) or 1.75 hours (recA- strain).
3. Chill the flask on ice for 5 min, transfer the culture into 50 ml centrifuge tube, and centrifuge it for 5 min at 4°C and 6,000 rpm to harvest cells. (After this stage, the cells should be kept cool throughout the cell preparation)
4. Remove the supernatant and suspend the cells with 1 ml of chilled 10% glycerol. Transfer the cell suspension into a 1.5 ml tube and centrifuge again for 1 min at 4°C and 12,000 rpm.
5. Remove the supernatant using a pipette and suspend the cells with 1 ml of chilled 10% glycerol. Centrifuge again for 1 min at 4°C and 12,000 rpm.
6. Remove the supernatant using a pipette and suspend the cells with 250 μl (depends on the amount of the cell) of chilled 10% glycerol.
Electroporation:
1. Mix 50 μl of the competent cell and DNA solution (the amount depends on the DNA plasmid) in a fresh 1.5 ml tube.
2. Transfer the mixture into chilled electroporation cuvette (2 mm gap), and pulse (Ec 2).
3. Add 1 ml of SOC immediately after the pulse.
4. Incubate it for 1 hour at 37°C.
5. Plate the culture on a selection plate, and incubate.
Gel Electrophoresis
Equipment:
1. Microwave
2. Electrophoresis comb
3. Electrophoresis trays
4. Pipette and pipette tips
5. Buffer tank
6. Voltage source
7. Glass bottle or Erlenmeyer flask
Consumables:
1. Agarose powder
2. TBE or TAE buffer
3. λ EH (λ DNA/EcoRI+HindIII) marker (2X) - Promega
4. 2X SLB or 10X SLB (sample loading buffer)
5. DNA sample
6. EtBr (Ethidium bromide)
7. RO (reverse osmosis)
Agarose gel preparation:
1. Adjust the amount of agarose to get the desired gel concentration (in this protocol, we’ll be using 0.7% agarose).
2. Weight 0.7 grams of agarose powder and add it to a glass bottle or Erlenmeyer flask.
3. Measure 100 ml of TBE buffer or TAE buffer and add it to the glass bottle or Erlenmeyer.
4. Put the mix into the microwave and heat it until the agarose is completely dissolved.
5. Let the mix cool down and pour it to the electrophoresis tray.
6. Set the electrophoresis comb and wait for the gel to solidify.
Sample preparation:
1. Samples are prepared in various ways depends on its proposes.
| Purpose | DNA Solution | SLB | Total Volume |
|---|---|---|---|
| Size checking | 2 μl | 10 μl (2X) | 12 μl |
| Gel extraction | depends on how much DNA solution to be extracted | 5 μl (10X) |
Agarose gel running:
1. When the agarose gel solidified, take out the electrophoresis comb.
2. Pour the TBE buffer or TAE buffer to the buffer tank. Note: buffer used for running has to be the same as the buffer used to prepare the gel solution.
3. Put the gel in the buffer tank.
4. Load 5 μl λ EH (λ DNA/EcoRI+HindIII) marker (2X) in a well and load the DNA sample mix with SLB in the remaining wells.
5. Run for 20 mins at 100V. Note: voltage and time varies.
Agarose gel visualization:
1. Take the gel out of the buffer tank.
2. Soak the gel in the EtBr solution for 8 mins and in RO for 30 secs.
3. Take the gel out of RO and visualize the DNA under UV transilluminator.
Gel Extraction and Purification
Equipment:
1. Eppendorf tubes
2. Collection tubes
3. Spin column
4. Pipette and pipette tips
5. Centrifuge
6. Scalpel
7. Dry Thermounit
Consumables:
1. Isopropanol
2. Binding buffer
3. Washing buffer
4. Elution buffer
Gel excision, solubilization and DNA binding:
1. Excise band with scalpel and transfer to a new eppendorf tube.
2. Weigh the gel slice in a tube (by measuring the weight difference of an empty eppendorf and the eppendorf with gel slice in the tube).
3. Add 3 volumes of binding buffer to 1 volume of gel (100 mg = 100 μl).
4. Incubate at 60°C for 2-10 mins (or until the gel has completely dissolved).
5. Add 1.5 volume of isopropanol and invert the eppendorf 10 times.
6. Place a spin column in a provided collection tube. Transfer 700 μl sample to the spin column and centrifuge for 30 secs, 12,000 rpm.
7. Discard flow-through and place the spin column back into the collection tube.
2. Wash:
1. Add 500 μl of washing buffer to the spin column and centrifuge for 30 secs, 12,000 rpm.
2. Discard flow-through and place the spin column back into the collection tube.
3. Add 200 μl of washing buffer to the spin column and centrifuge for 5 mins, 12,000 rpm.
4. Discard flow-through and place the spin column back into the collection tube.
3. DNA elution:
1. Transfer spin column to clean eppendorf. Add 50 μl of elution buffer to the spin column. Centrifuge for 2 mins, 12,000 rpm.
2. Collect the pure sample in the eppendorf and discard the spin column.
Keep the DNA solution in -20°C freezer.
Ligation
Equipment:
1. Eppendorf tubes
2. Pipette and pipette tips
3. Centrifuge
Consumables:
1. T4 DNA ligase (NEB)
2. T4 DNA ligase buffer (NEB)
1. Set up the following reaction on ice:
| Vector | 12 μl |
| Insert | 40 μl |
| T4 DNA ligase buffer (10x) | 5.8 μl |
| T4 DNA ligase | 0.5 μl |
| Total Volume | 58.3 μl |
|---|
2. Gently mix the reaction and centrifuge.
3. Incubate at 16°C overnight.
4. Keep the DNA solution in -20°C freezer.
PCR Clean-up
Equipment:
1. Eppendorf tubes
2. Collection tubes
3. Spin column
4. Pipette and pipette tips
5. Centrifuge
Consumables:
1. Isopropanol
2. Binding buffer
3. Washing buffer
4. Elution buffer
5. MQ or ddH2O
DNA binding:
1. Add MQ or ddH2O to DNA solution until the volume reaches 100 μl.
2. Add 300 μl of binding buffer to the solution.
3. Add 150 μl of isopropanol and invert the eppendorf 10 times.
4. Place a spin column in a provided collection tube. Transfer 550 μl sample to the spin column and centrifuge for 30 secs, 12,000 rpm.
5. Discard flow-through and place the spin column back into the collection tube.
Wash
1. Add 500 μl of washing buffer to the spin column and centrifuge for 30 secs, 12,000 rpm.
2. Discard flow-through and place the spin column back into the collection tube.
3. Add 200 μl of washing buffer to the spin column and centrifuge for 5 mins, 12,000 rpm.
4. Discard flow-through and place the spin column back into the collection tube.
DNA elution
1. Transfer spin column to clean eppendorf. Add 50 μl of elution buffer to the spin column. Centrifuge for 2 mins, 12,000 rpm.
2. Collect the pure sample in the eppendorf and discard the spin column.
3. Keep the DNA solution in -20°C freezer.
Plasmid Isolation: Miniprep
Equipment:
1. Eppendorf tubes
2. Collection tubes
3. Spin column
4. Pipette and pipette tips
5. Centrifuge
6. Ice bucket
Consumables:
1. Suspension buffer
2. Lysis buffer
3. Binding buffer
4. Washing buffer I
5. Washing buffer II
6. Elution buffer
7. LB broth
8. Antibiotic as needed
1. Prepare 2-5 ml overnight culture in LB medium with the antibiotic (depends on the plasmid to be extracted).
2. Collect cells from the overnight culture with a centrifuge (12,000 rpm 30 sec) and discard the supernatant.
3. Add 250 μl of suspension buffer and suspend bacterial cells by vortex.
4. Add 250 μl of lysis buffer and mix gently by inverting the tube 10 times.
5. Add 350 μl of binding buffer and mix gently by inverting the tube 10 times.
6. Incubate on ice for 5 mins.
7. Centrifuge at 4°C, 12,000 rpm for 10 mins.
8. Place a spin column in a provided collection tube. Transfer 750 μl of supernatant to spin column.
9. Centrifuge at room temperature, 12,000 rpm for 30 sec and discard the flow-through.
10. Add 500 μl of washing buffer I to the spin column and centrifuge for 30 sec. Discard the flow-through.
Restriction Enzyme Digestion
| Restriction Enzymes | Buffer (Buffer will depend on restriction enzyme being used) |
|---|---|
| EcoRI | SuR E/Cut Buffer H (10X) - Roche |
| XbaI | CutSmart Reaction Buffer (10x) - NEB or 3.1 - NEB |
| SpeI | CutSmart Reaction Buffer (10x) - NEB |
| PstI | 3.1 (10X) - NEB |
Equipment:
1. Eppendorf tubes
2. Pipette and pipette tips
3. Ice bucket
4. Incubator (37°C)
Consumables:
1. Restriction enzymes
2. Buffer 10x (depends on its restriction enzymes)
3. DNA sample
4. MQ od ddH2O (for single digestion)
1. On the ice, add all the components.
Double Digestion:
| DNA | 43 μl |
| Buffer 10x | 5 μl |
| Restriction enzyme 1 | 1 μl |
| Restriction enzyme 2 | 1 μl |
| Total Volume | 50 μl |
|---|
Single Digestion (Structure Check):
| Plasmid DNA | 2 μl |
| Buffer 10x | 1.5 μl |
| Restriction enzyme | 0.5 μl |
| MQ or ddH2O | 11 μl |
| Total Volume | 15 μl |
|---|
2.Mix gently and incubate for 1-2 hours for double digestion or 30 mins - 1 hour for single digestion at 37°C. Note: Incubation time varies along the total volume of the reaction.
SDS-PAGE
Equipment:
1. Eppendorf tubes
2. Pipette and pipette tips
3. Centrifuge
4. Vortex
5. Ice bucket
6. Empty box
7. Dry Thermounit
8. Glass plates
9. 10-well comb
10. Spacer
11. Clamp
12. Casting stand
13. Buffer tank
14. Voltage source
15. Shaker
Consumables:
1. H2O
2. 30% acrylamide
3. 1.5M tris pH 8.8
4. 1M tris pH 6.8
5. 10% SDS
6. 10% APS
7. TEMED
8. Prestained Protein Marker - Bioman
9. Protein dye
10. Overnight culture (sample)
11. CBB (Coomassie Brilliant Blue) staining solution
12. Destaining buffer
13. Tank buffer (1X)
14. Iso-propanol
Resolving gel preparation
1. Adjust the amount of agarose to get the desired gel concentration
2. In a 50 ml eppendorf tube add all the components 15% gel concentration
| Total Volume | 5 ml | 10 ml | 15 ml |
|---|---|---|---|
| H2O (ml) | 1.15 | 2.3 | 3.4 |
| 30% acrylamide mix (ml) | 2.5 | 5 | 7.5 |
| 1.5M Tris pH 8.8 (ml) | 1.25 | 2.5 | 3.3 |
| 10% SDS (ml) | 0.05 | 0.1 | 0.15 |
| 10% APS (ml) | 0.10 | 0.2 | 0.3 |
| TEMED (ml) | 0.002 | 0.004 | 0.006 |
3. Pour the mixture in between the glasses and add iso-propanol afterward.
4. Wait for 10-15 mins or until the gel solidifies.
5. Pour out the iso-propanol.
Stacking gel preparation
1. In a 50 ml eppendorf tube add all the components
| Total Volume | 1.5 ml | 3 ml | 5 ml |
|---|---|---|---|
| H2O (ml) | 1.05 | 2.1 | 3.4 |
| 30% acrylamide mix (ml) | 0.25 | 0.5 | 0.83 |
| 1M Tris pH 6.8 (ml) | 0.19 | 0.38 | 0.63 |
| 10% SDS (ml) | 0.015 | 0.03 | 0.05 |
| 10% APS (ml) | 0.03 | 0.06 | 0.05 |
| TEMED (ml) | 0.0015 | 0.003 | 0.005 |
2. Pour the mixture in between the glass plates and add the 10-well comb.
3. Wait for 10-15 mins or until the gel solidifies.
Sample preparation
1. Take overnight cultures with the desired volume and centrifuge.
2. Take 12 μl of pellet or supernatant (depends on necessity) and move to a new fresh eppendorf.
3. Add 3 μl dye into the eppendorf. Vortex and centrifuge briefly.
4. Heat the eppendorf at 100°C for 10 mins and centrifuge briefly.
5. Put samples in the ice bucket.
Gel running
1. When the gel solidified, set up the running equipment and pour 1X tank buffer.
2. Take out the comb and load 3 μl of the marker into a well and load the samples into the remaining wells.
3. Run the first 30 mins at 120V then continue to run for 1 hour at 150 V. Note: voltage and time may vary.
Staining
1. Take out the glass plates out of the buffer tank and split up the glass plates to take out the gel.
2. the stacking gel and put the resolving gel inside an empty box, then add the CBB staining solution until it covers the gel.
Put the box on a shaker and shake for 30 mins or until the gel turns blue.
Destaining
1. Pour the CBB staining solution back to the bottle and add the destaining buffer into the box until it covers the gel.
2. Put the box on a shaker and shake until the protein bands are visible or until the gel turns white.
3. Pour out the destaining buffer.
Standard PCR
Equipment:
1. PCR tubes
2. Ice bucket
3. Thermocycler
4. Pipette and pipette tips
Consumables:
1. MQ or ddH2O
2. 2x PCR buffer for KOD FX
3. dNTPs 2 mM
4. Forward and reverse primer (10 μM)
5. KOD FX polymerase
1. On the ice, add all components in a PCR tube, making up to 50 µl volume reaction.
| Components | Volume (μl) |
|---|---|
| MQ or ddH2O | 1.1 |
| 10x PCR buffer | 1 |
| dNTP 2.5 mM | 0.8 |
| Template | 1 |
| Forward primer 10 μM | 1 |
| Reverse primer 10 μM | 0.1 |
| Taq polymerase + vent | 0.1 |
| Total Volume | 10 |
2. Gently mix the PCR reactions and centrifuge briefly.
3. Transfer the PCR tubes to a thermocycler.
| Step | Temperature | Time |
|---|---|---|
| Initial denaturalization | 94°C | 2 mins |
| 25 - 35 cycles | 98°C (denaturation) | 30 secs |
| 55°C (annealing) | 30 secs | |
| 68°C (extension) | 2 mins | |
| Final extension | 68°C | 2 mins |
| Hold | 16°C (holding for a short time) or 4°C (holding for a long time) | ∞ |
Media Preparation
1.LB (Lysogeny Broth)
Equipment:
1. Glass bottle (for LB broth) or Erlenmeyer flask (for agar medium)
2. Magnetic stirrer
3. Magnetic stirring bar
4. Plastic measure jug
5. Measure cylinder
6. Spoon
7. Petri dish (for agar medium)
8. Aluminum foil
9. Electronic balance
10. Weighing paper or weighing bowl
11. Pipette and pipette tips
Consumables:
1. DW (distilled water)
2. Tryptone
3. Yeast extract
4. NaCl
5. NaOH (10N)
6. Agar (for LB agar)
7. Antibiotics (for selection plate)
1. Prepare mixture as the following inside a jar with a magnetic stirring bar inside and place it on the magnetic stirrer.
| DW | 98 ml |
| Tryptone | 1 g |
| Yeast Extract | 0.5 g |
| NaCl | 1 g |
| NaOH (10N) | 20 μl |
| Total Volume | 100 ml |
|---|---|
| Agar (for LB agar) | 1.5% |
2. Mix well, pour the mixture into the bottle or flask and autoclave. Notes: For agar medium, pour the mixture to the petri dish and dry the plates.
For selection plates, add antibiotics as the following before pouring the mixture to the petri dish:
2.SOB (Super Optimal Broth)
Equipment:
1. Flask
2. Magnetic stirrer
3. Magnetic stirring bar
4. Plastic measure jug
5. Measure cylinder
6. Spoon
7. Aluminum foil
8. Electronic balance
9. Weighing paper or weighing bowl
10. Pipette and pipette tips
Consumables:
1. DW (distilled water)
2. Tryptone
3. Yeast extract
4. 5M NaCl
5. 3M KCl
1. Prepare mixture as the following inside a jar with a magnetic stirring bar inside and place it on the magnetic stirrer.
| DW | 96 ml |
| Tryptone | 2 g |
| Yeast Extract | 0.5 g |
| 5M NaCl | 0.2 ml |
| 3M KCL | 83 μl |
| Total Volume | 100 ml |
|---|
2. Mix well, pour the mixture into the flask and autoclave.
3.SOC
Equipment:
1. Pipette and pipette tips
Consumables:
1. SOB medium in a glass bottle
2. 1M MgCl2
3. 1M MgSO4
4. 20% glucose
1. Add chemicals as the following to a bottle of 100 ml SOB (autoclaved)
| 1M MgCl2 | 96 ml |
| 1M MgSO4 | 2 g |
| 20% glucose | 0.5 g |
4. M9 Medium
Equipment:
1. Pipette and pipette tips
Consumables:
1. M9 salts (5x)
2. 20% glucose
3. 1M MgSO4
4. 1M CaCl2
5. H2O
1. Add chemicals as the following to a bottle
| M9 salts (5X) | 20 ml |
| 20% glucose | 2 ml |
| 1M MgSO4 | 200 μl |
| 1M CaCl2 | 10 μl |
| H2O | 78 ml |
| Total Volume | 100 ml |
|---|
2. Mix well, pour the mixture into the flask and autoclave.
Bubble Production
Equipment:
1. Beaker
2. Hot plate magnetic heater
3. Weighing scale
4. Pipette and pipette tips
5. Centrifuge tubes
Consumables:
1. 1 g sodium alginate (mixed with 100 ml ddH2O, heated and mixed using a magnetic stirrer until no powder precipitate is left)
2. MQ or ddH2O
3. 1.5 g CaCl2 (mixed with 100 ml ddH2O)
4. 15.6 g NaH2PO4 (mixed with 200 ml ddH2O, then divided into 16 tubes each containing 12.5 ml)
5. 2 ml freshly cultured bacteria (OD600= 1)
1. Prepare freshly cultured bacteria.
2. Adjust the OD600 of the bacteria to 1.
3. Pour 2 ml of the bacteria into 50 ml sodium alginate, and then mix well.
4. Using a pipette, slowly drip 2.5 ml of the sodium alginate solution drop by drop into 100 mL CaCl2.
5. Each experiment is supposed to accomodate around 7.7 x 107 cells of bacteria.
6. Separate the bubbles from the CaCl2 solution using filtration or decantation.
7. Pour the bubbles into 12.525 ml of buffer, and incubate for 2 hours before plating in plate medium.
Oxidative Assay
Equipment:
1. 96 wells
2. Pipette and pipette tips
3. Plate reader
Consumables:
1. Overnight culture (sample)
2. Paraquat (PQ)
3. Hydrogen peroxide (H2O2)
4. MQ or ddH2O
1. Apply 180 μl culture in 96 wells and measure OD600 and sfGFP expression as control value.
2. After 1.5 hour, dilute PQ and H2O2 with ddH2O and add it into the well as inducer.
3. Measure the OD600 and sfGFP expression through plate reader every hour.
4. Repeat Step 3 six times.
Functional Test(in vitro)
Equipment:
1. Eppendorf tubes
2. Pipette and pipette tips
3. High power press cell disruptor
4. 37°C incubator
Consumables:
1. 12 h cultured bacteria (JJU, CoaBC)
2. 50 mM L-Cysteine
3. 20 mM PLP
4. PBS (pH 7.4 buffer)
1. Wash bacteria twice.
2. Adjust the OD600 of bacteria to 10 and retrieve the supernatant after lysing the bacteria.
3. Run SDS-PAGE on the supernatant and whole cell samples.
4. Add a premix solution to eppendorf tubes, the contents of the premix is shown in the table below.
| Components | Volume(μl) | |||
| 50mM L-Cysteiene | 20 | |||
| JJU | 80 | 107 | 53 | respectively 1:1/2:1/1:2 |
| CoaBC | 80 | 53 | 107 | |
| 20mM PLP | 5 | |||
| PBS (pH 7.4 buffer) | 15 | |||
| Total Volume | 200 | |||
|---|---|---|---|---|
5. Eppendorf tubes are incubated at 37oC for 1 hour.
6. After incubation, eppendorf tubes is centrifuged at 12500 rpm for 5 minutes.
7. Rtrieve the supernatant to conduct HPLC, to measure taurine concentration.
Functional Test(in vivo)
Equipment:
1. Eppendorf tubes
2. Pipette and pipette tips
3. High power press cell disruptor
4. 37°C incubator
Consumables:
1. LB
2. 50 mM L-Cysteine
3. 20 mM PLP
4. IPTG
1. Preculture 4 ml LB with sample at 37°C overnight.
2. From preculture, take 2% of the bacterial culture to a fresh 20 ml LB culture.
3. Test OD600 per 2 h to determine the growth curve of the bacteria.
4. When OD600 is around 0.6, add in 20 μl IPTG as an inducer and then add L-Cystine and PLP for chemical reaction.
5. Collect samples at set hours.
6. To determine extracellular taurine concentration, centrifuge the sample and take supernatant for HPLC.
7. To determine intracellular taurine concentration, lysis the bacteria, centrifuge it then take the supernatant to conduct HPLC.
8. Run SDS-PAGE to confirm the protein expression.
