Team:NCKU Tainan/Protocols
This page is about our protocols, you can find all the experimental process you need.
Enjoy!
Bacterial Glycerol Stock
Equipment:
1. Pipette and pipette tips
2. Cryovial
Consumables:
1. 50% glycerol (autoclaved)
2. Bacterial overnight culture with an antibiotic if necessary
1. Put 1 ml of bacterial overnight culture into the cryovial and add 500 μl of 50% glycerol. Mix it well.
2. Keep it in the -80°C freezer.
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 2kbp/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 | CusSmart 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 | Temp | 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.
Functional Test
can gene kill switch functional test
Equipment:
1. CO2 incubator
2. Toothpick
Consumables:
1. LB plates (can also with the desired antibiotic)
1. Divide the plate into several parts depends on how many colonies are being tested.
2. Streak the plates using the toothpick.
3. Grow the bacteria in the 37°C CO2 incubator and normal incubator for comparison.
dapA gene kill switch functional test
Equipment:
1. 37°C incubator
2. Toothpick
Consumables:
1. LB plates (can also with the desired antibiotic)
2. LB + DAP plates (concentration of DAP may vary)
1. Divide the plates with DAP chemical and without DAP chemical into several parts depends on how many colonies are being tested.
2. Streak the plates using the toothpick.
3. Grow the bacteria in the 37°C incubator.
p-Cresol Sensing Functional Test
Equipment:
1. 96 well-plate
2. Pipette and tips
3. 37°C incubator
4. Spectrofluorometer
Consumables:
1. Bacterial culture
2. p-Cresol
1. Make overnight cultures induced with different concentrations of p-Cresol and also without p-Cresol for control. Incubate at 37°C incubator.
2. Load 200 μl of samples onto the 96 well plates. Arrange with your personal preference.
3. Measure fluorescence using the spectrofluorometer.
pFNR-GFP Characterization Assay
Equipment:
1. Black 96 well-plate
2. Pipette and tips
3. 37°C incubator
4. 37°C anaerobic incubator
5. Test tube (glass)
6. Cell density reader
7. Fluorescence reader
Consumables:
1. Bacterial culture (E. coli DH5a with pFNR-GFP and E. coli DH5a with pSB1C3)
2. Eppendorf
3. LB medium with appropriate antibiotic
1. Grow bacterial culture with 4 ml LB and appropriate antibiotics, put it in 37°C anaerobic incubator and also normal incubator overnight. For more accurate data, make 3 cultures for every strain.
2. Refresh all bacterial culture in 10 ml LB with appropriate antibiotics.
3. Take 1 ml bacterial culture into Eppendorf every 2 hours for measurement.
4. Add 200 μl of bacterial culture to the 96-well plate. Set up the fluorescence reader (in our case, wtgfp: excitation-395 nm, emission-509 nm) and measure the fluorescence.
Spot on Lawn Assay
Equipment:
1. Pipette and tips
2. -80°C fridge
3. 37°C anaerobic incubator
Consumables:
1. Bacterial culture
2. BHI plate
3. 0.22 μm filter
4. Purified bacteriocin (positive control)
5. LB medium
1. Grow overnight cultures of c. difficile and bacteria carrying plasmid with bacteriocin in 6 ml LB.
2. Take 100 μl of the c. difficile overnight culture and streak evenly on the dried BHI plate.
3. To test the secretion ability of bacteriocin, take 2 ml overnight culture of bacteriocin producing bacteria and filter it through a 0.22 μm filter. Spot 5 μl of the filtered solution on the streaked BHI plate.
4. To test the expression of bacteriocin, take 2 ml overnight culture of bacteriocin producing bacteria and put it in -80°C fridge. Freeze for 10 min and take the frozen bacteria back to the room temperature to unfroze it. Repeat this step twice. Then, take the culture solution and filter it through a 0.22 μm filter and spot 5 μl of the filtered solution on the streaked BHI plate.
5. For positive control, purified bacteriocin without yebF secretion tag kindly proved by one of the PI’s lab are used.
6. Grow the plate in the anaerobic incubator and observe the inhibition zone.
TAL Functional Test
Equipment:
1. 37°C anaerobic incubator
2. Pipette and tips
3. Spectrophotometer (NanoDrop)
Consumables:
Bacterial culture
1. n-octanol
2. LB
3. LB + 2 mM tyrosine
4. LB + 1 mM tyrosine
5. LB + 500 μM tyrosine
6. p-Coumaric acid
7. Lysis buffer
8. Neutralization buffer
p-Coumaric acid fermentation:
1. For each E.coli strain, bacterial colony is inoculate to 6 ml LB medium containing appropriate antibiotic and grow overnight in normal 37°C incubator.
2. Incubate for 16 hours.
3. Take 60 μl of bacterial culture to refresh in 6 ml LB containing containing appropriate antibiotic and different concentration of tyrosine.
4. Incubate the refreshed cell culture in anaerobic incubator at 37°C.
5. Samples are collected in the 24- and 48- hour, after the initiation of the anaerobic 37°C incubation.
Sample collection:
1. 250 μl of samples are collected at the 2 particular times stated above.
2. The positive control is prepared by collecting 225 μl of negative control (Wild-type strain) culture with 25 μl of 500 μM, 250 μM and 50 μM of p-Coumaric acid in LB to create a bacterial culture with p-Coumaric acid concentration of 50 μM, 25 μM and 5 μM.
Sample Extraction:
1. Directly after sample collection, the samples are lysed with 25 μl of Lysis buffer (PD2) and neutralized with 43.5 μl neutralization buffer (PD3). (Thermo plasmid MiniPrep)
2. All samples are acidified in 12.5 μl concentrated acetic acid and vortexed.
3. Samples are then induced with 50 μl n-octanol (the organic phase in two-phase extraction).
4. Vortexing and centrifugation (>12000 g, 1 min) followed and the upper organic phase is collected for measurement.
Measurement:
1. The absorbance in the UV-spectrum (190 nm-350 nm) of the octanol extracts is measured in a spectrophotometer (NanoDrop).
2. The spectrophotometer blanked by pure n-octanol.
3. All measurements are done on 2 μl of extractant.
4. Absorbance measurements are done in the same way on the standards of p-Coumaric acid in octanol prepared from solid p-Coumaric acid.
